bluuebunny/crossref_metadata_2025 huggingface dataset:
Response to caffeine and ryanodine receptor isoforms in mouse skeletal muscles
Authors: Rossi R. , Bottinelli R. , Sorrentino V. , Reggiani C.
Published: December 2017

The response to caffeine was studied in mouse muscles [diaphragm, soleus, and extensor digitorum longus (EDL)] with different ryanodine receptor isoform (RyR1, RyR3) composition and in single permeabilized muscle fibers dissected from diaphragm of wild-type (WT) and RyR3-deficient (RyR3−/−) mice at 1, 15, 30, and 60 postnatal days (PND). The caffeine response decreased during development, and, in adult mice, was greater in diaphragm, lower in EDL, and intermediate in soleus. This suggests a direct relation between response to caffeine and RyR3 expression. The lack of RyR3 reduced caffeine response in young, but not in adult mice, and did not abolish the age-dependent variation and the intermuscle differences. In diaphragm single fibers, the response to caffeine increased during development and was reduced in fibers lacking RyR3 both at 15 and 60 PND. A population of fibers highly responsive to caffeine was present in adult WT and disappeared in RyR3−/−. The results confirm the contribution of RyR3 to calcium release for contractile response and clarify the contribution of RyR3 to developmental changes and intermuscle differences.
Rabbit conjunctival epithelium transports fluid, and P2Y22 receptor agonists stimulate Cl− and fluid secretion
Authors: Li Yansui , Kuang Kunyan , Yerxa Benjamin , Wen Quan , Rosskothen Heinz , Fischbarg Jorge
Published: December 2017

Rabbit conjunctival epithelium exhibits UTP-dependent Cl− secretion into the tears. We investigated whether fluid secretion also takes place. Short-circuit current ( I sc) was 14.9 ± 1.4 μA/cm2( n = 16). Four P2Y2 purinergic receptor agonists [UTP and the novel compounds INS365, INS306, and INS440 (Inspire Pharmaceuticals)] added apically (10 μM) resulted in temporary (∼30 min) I sc increases (88%, 66%, 57%, and 28%, respectively; n = 4 each). Importantly, the conjunctiva transported fluid from serosa to mucosa at a rate of 6.5 ± 0.7 μl · h−1 · cm−2 (range 2.1–15.3, n = 20). Fluid transport was stimulated by mucosal additions of 10 μM: 1) UTP, from 7.4 ± 2.3 to 10.7 ± 3.3 μl · h−1 · cm−2, n = 5; and 2) INS365, from 6.3 ± 1.0 to 9.8 ± 2.5 μl · h−1 · cm−2, n = 5. Fluid transport was abolished by 1 mM ouabain ( n = 5) and was drastically inhibited by 300 μM quinidine (from 6.4 ± 1.2 to 3.6 ± 1.0 μl · h−1 · cm−2, n = 4). We conclude that this epithelium secretes fluid actively and that P2Y2 agonists stimulate both Cl− and fluid secretions.
Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits
Authors: Blazer-Yost Bonnie L. , Butterworth Michael , Hartman Amy D. , Parker Gretchen E. , Faletti Carla J. , Els Willem J. , Rhodes Simon J.
Published: December 2017

A6 model renal epithelial cells were stably transfected with enhanced green fluorescent protein (EGFP)-tagged α- or β-subunits of the epithelial Na+channel (ENaC). Transfected RNA and proteins were both expressed in low abundance, similar to the endogenous levels of ENaC in native cells. In living cells, laser scanning confocal microscopy revealed a predominately subapical distribution of EGFP-labeled subunits, suggesting a readily accessible pool of subunits available to participate in Na+ transport. The basal level of Na+ transport in the clonal lines was enhanced two- to fourfold relative to the parent line. Natriferic responses to insulin or aldosterone were similar in magnitude to the parent line, while forskolin-stimulated Na+ transport was 64% greater than control in both the α- and β-transfected lines. In response to forskolin, EGFP-labeled channel subunits traffic to the apical membrane. These data suggest that channel regulators, not the channel per se, form the rate-limiting step in response to insulin or aldosterone stimulation, while the number of channel subunits is important for basal as well as cAMP-stimulated Na+transport.
Involvement of DMT1 in uptake of Cd in MDCK cells: role of protein kinase C
Authors: Olivi Luisa , Sisk Jeanne , Bressler Joseph
Published: December 2017

The involvement of iron (Fe) transporters in the uptake of cadmium (Cd) was examined in Madin-Darby kidney cells (MDCK). The uptake of Cd displayed properties that are associated with the Fe transporter divalent metal transporter 1 (DMT1). For example, the uptake of Cd and Fe was reduced by altering the cell membrane potential. The uptake of Cd was blocked by Fe, and the uptake of Fe was blocked by Cd. Also, the uptake of Cd and Fe was higher in MDCK cells bathed in a buffer at low pH. Increased uptake of Fe and Cd was observed in the HEK-293 cell line overexpressing DMT1. Overnight treatment of MDCK cells with the protein kinase C activator phorbol 12,13-dibutyrate (PDBu) resulted in increased uptake of Cd and Fe and an increase in DMT1 mRNA. An increase in newly transcribed DMT1 mRNA was not observed, suggesting that PDBu does not increase DMT1 mRNA by activating transcription. Rather, the increase was most likely due to greater stability of DMT1 mRNA, because the rate of degradation of DMT1 mRNA was slower in MDCK cells treated with PDBu. Our results suggest that Fe and Cd are transported in MDCK cells by a transporter with biochemical properties similar to those of DMT1.
Polyamine depletion stabilizes p53 resulting in inhibition of normal intestinal epithelial cell proliferation
Authors: Li Li , Rao Jaladanki N. , Guo Xin , Liu Lan , Santora Rachel , Bass Barbara L. , Wang Jian-Ying
Published: December 2017

The p53 nuclear phosphoprotein plays a critical role in transcriptional regulation of target genes involved in growth arrest and apoptosis. The natural polyamines, including spermidine, spermine, and their precursor putrescine, are required for cell proliferation, and decreasing cellular polyamines inhibits growth of the small intestinal mucosa. In the current study, we investigated the mechanisms of regulation of p53 gene expression by cellular polyamines and further determined the role of the gene product in the process of growth inhibition after polyamine depletion. Studies were conducted both in vivo and in vitro using rats and the IEC-6 cell line, derived from rat small intestinal crypt cells. Levels for p53 mRNA and protein, transcription and posttranscription of the p53 gene, and cell growth were examined. Depletion of cellular polyamines by treatment with α-difluoromethylornithine (DFMO) increased p53 gene expression and caused growth inhibition in the intact small intestinal mucosa and the cultured cells. Polyamine depletion dramatically increased the stability of p53 mRNA as measured by the mRNA half-life but had no effect on p53 gene transcription in IEC-6 cells. Induction of p53 mRNA levels in DFMO-treated cells was paralleled by an increase in the rate of newly synthesized p53 protein. The stability of p53 protein was also increased after polyamine depletion, which was associated with a decrease in Mdm2 expression. When polyamine-deficient cells were exposed to exogenous spermidine, a decrease in p53 gene expression preceded an increase in cellular DNA synthesis. Inhibition of the p53 gene expression by using p53 antisense oligodeoxyribonucleotides significantly promoted cell growth in the presence of DFMO. These findings indicate that polyamines downregulate p53 gene expression posttranscriptionally and that growth inhibition of small intestinal mucosa after polyamine depletion is mediated, at least partially, through the activation of p53 gene.
Common mechanisms of monoacylglycerol and fatty acid uptake by human intestinal Caco-2 cells
Authors: Ho Shiu-Ying , Storch Judith
Published: December 2017

Free fatty acids (FFA) and sn-2-monoacylglycerol ( sn-2-MG), the two hydrolysis products of dietary triacylglycerol, are absorbed from the lumen into polarized enterocytes that line the small intestine. Intensive studies regarding FFA transport across the brush-border membrane of the enterocyte are available; however, little is known about sn-2-MG transport. We therefore studied the kinetics of sn-2-MG transport, compared with those of long-chain FFA (LCFA), by human intestinal Caco-2 cells. To mimic postprandial luminal and plasma environments, we examined the uptake of taurocholate-mixed lipids and albumin-bound lipids at the apical (AP) and basolateral (BL) surfaces of Caco-2 cells, respectively. The results demonstrate that the uptake of sn-2-monoolein at both the AP and BL membranes appears to be a saturable function of the monomer concentration of sn-2-monoolein. Furthermore, trypsin preincubation inhibits sn-2-monoolein uptake at both AP and BL poles of cells. These results suggest that sn-2-monoolein uptake may be a protein-mediated process. Competition studies also support a protein-mediated mechanism and indicate that LCFA and LCMG may compete through the same membrane protein(s) at the AP surface of Caco-2 cells. The plasma membrane fatty acid-binding protein (FABPpm) is known to be expressed in Caco-2, and here we demonstrate that fatty acid transport protein (FATP) is also expressed. These putative plasma membrane LCFA transporters may be involved in the uptake of sn-2-monoolein into Caco-2 cells.
Essential role of ICAM-1 in mediating monocyte adhesion to aortic endothelial cells
Authors: Kevil Christopher G. , Patel Rakesh P. , Bullard Daniel C.
Published: December 2017

Monocyte-endothelial cell interactions have been implicated in the pathogenesis of a number of vascular diseases that target arterial and aortic endothelium, including atherosclerosis. Many different adhesion molecules, such as intercellular adhesion molecule (ICAM)-1, are thought to mediate monocyte binding to endothelial cells during the development of these diseases. However, conflicting results have been reported regarding the specific role of ICAM-1 in these events. In this study, we used a genetic approach to determine the contribution of ICAM-1 in mediating monocyte adhesion to mouse aortic endothelial cells (MAEC) derived from both wild-type and ICAM-1−/− mice. Treatment of wild-type MAEC with oxidized low-density lipoprotein significantly induced both WEHI 274.1 and whole blood monocyte adhesion, whereas similarly treated ICAM-1−/− MAEC showed a complete inhibition of monocyte binding. Dose-response treatment with tumor necrosis factor-α also increased monocyte adhesion to wild-type MAEC, but significant adhesion was only observed at higher doses for ICAM-1−/− MAEC. These data demonstrate a crucial role for ICAM-1-mediated monocyte-endothelial cell interactions in response to specific stimuli involved in inflammatory vascular diseases.
Opposing gates model for voltage gating of gap junction channels
Authors: Chen-Izu Ye , Moreno Alonso P. , Spangler Robert A.
Published: December 2017

Gap junctions are intercellular channels that link the cytoplasm of neighboring cells. Because a gap junction channel is composed of two connexons docking head-to-head with each other, the channel voltage-gating profile is symmetrical for homotypic channels made of two identical connexons (hemichannels) and asymmetric for the heterotypic channels made of two different connexons (i.e., different connexin composition). In this study we have developed a gating model that allows quantitative characterization of the voltage gating of homotypic and heterotypic channels. This model differs from the present model in use by integrating, rather than separating, the contributions of the voltage gates of the two member connexons. The gating profile can now be fitted over the entire voltage range, eliminating the previous need for data splicing and fusion of two hemichannel descriptions, which is problematic when dealing with heterotypic channels. This model also provides a practical formula to render quantitative several previously qualitative concepts, including a similarity principle for matching a voltage gate to its host connexon, assignment of gating polarity to a connexon, and the effect of docking interactions between two member connexons in an intact gap junction channel.
Skeletal muscle cell hypertrophy induced by inhibitors of metalloproteases; myostatin as a potential mediator
Authors: Huet Clotilde , Li Zhi-Fang , Liu Hai-Zhen , Black Roy A. , Galliano Marie-Florence , Engvall Eva
Published: December 2017

Cell growth and differentiation are controlled in many tissues by paracrine factors, which often require proteolytic processing for activation. Metalloproteases of the metzincin family, such as matrix metalloproteases and ADAMs, recently have been shown to be involved in the shedding of growth factors, cytokines, and receptors. In the present study, we show that hydroxamate-based inhibitors of metalloproteases (HIMPs), such as TAPI and BB-3103, increase the fusion of C2C12 myoblasts and provoke myotube hypertrophy. HIMPs did not seem to effect hypertrophy via proteins that have previously been shown to regulate muscle growth in vitro, such as insulin-like growth factor-I, calcineurin, and tumor necrosis factor-α. Instead, the proteolytic maturation of myostatin (growth differentiation factor-8) seemed to be reduced in C2C12 cells treated with HIMPs, as suggested by the presence of nonprocessed myostatin precursor only in hypertrophic myotubes. Myostatin is a known negative regulator of skeletal muscle growth, belonging to the transforming growth factor-β/bone morphogenetic protein superfamily. These results indicate that metalloproteases are involved in the regulation of skeletal muscle growth and differentiation, that the proteolytic maturation of myostatin in C2C12 cells may be directly or indirectly linked to the activity of some unidentified HIMP-sensitive metalloproteases, and that the lack of myostatin processing on HIMP treatment may be a mediator of myotube hypertrophy in this in vitro model.
Sustained hyposmotic stress induces cell death: apoptosis by defeat
Authors: Jäckle Tina , Hasel Cornelia , Melzner Ingo , Brüderlein S. , Jehle Peter M. , Möller Peter
Published: December 2017

We describe sustained hyposmotic stress as a novel type of environmental condition enforcing apoptosis. In a dose- and time-dependent fashion, hyposmotic stress leads to a delayed type of apoptosis with considerable variations in constitutive sensitivity among different cell types. For example, after 48 h at 84 mosmol/l, the death rate ranged from 10.8 ± 0.7% in AsPc1 human pancreatic carcinoma cells to 72.0 ± 1.6% in HK-2 human kidney tubule cells. Caspase inhibitors rendered cells more resistant to hyposmolar stress; the caspase 3 inhibitor Ac-Asp-Glu-Val-aspartic acid aldehyde was the most efficient. After 24 h of stress, HT-29 colon carcinoma and HK-2 cells had increased their mitochondrial mass. This went along with an increase in mitochondrial membrane potential in HT-29 cells but with a decrease in HK-2 cells. Starting at 2 h of stress, we detected transient CD95L transcription followed by surface expression of CD95L in HT-29 but not in HK-2 cells. Inhibitory CD95L antibody partially inhibited specific death in HT-29 but not in HK-2 cells. Thus, as in other types of stress-induced apoptosis, the CD95/CD95L system is one of the different routes to suicide optionally used by hyposmotically stressed cells. Our findings may have clinical implications for the prevention and treatment of tissue damage caused by severe hyposmolar states.
Lung epithelial barrier function and wound healing are decreased by IL-4 and IL-13 and enhanced by IFN-γ
Authors: Ahdieh Minoo , Vandenbos Tim , Youakim Adel
Published: December 2017

To understand the effects of cytokines on epithelial cells in asthma, we have investigated the effects of interleukin (IL)-4, IL-13, and interferon (IFN)-γ on barrier function and wound healing in Calu-3 human lung epithelial cells. IL-4 and IL-13 treatment of Calu-3 cells grown on Transwell filters resulted in a 70–75% decrease in barrier function as assessed by electrophysiological and [14C]mannitol flux measurements. In contrast, IFN-γ enhanced barrier function threefold using these same parameters. Cells treated concurrently with IFN-γ and IL-4 or IL-13 showed an initial decline in barrier function that was reversed within 2 days, resulting in barrier levels comparable to control cells. Analysis of the tight junction-associated proteins ZO-1 and occludin showed that IL-4 and IL-13 significantly reduced ZO-1 expression and modestly decreased occludin expression compared with controls. IFN-γ, quite unexpectedly given its enhancing effect on barrier function, reduced expression of ZO-1 and occludin to almost undetectable levels compared with controls. In wound-healing assays of cells grown on collagen I, IL-4 and IL-13 decreased migration, whereas IFN-γ treatment enhanced migration, compared with control cells. Addition of IFN-γ, in combination with IL-4 or IL-13, restored migration of cells to control levels. Migration differences observed between the various cytokine treatments was correlated with expression of the collagen I-binding α2β1-integrin at the leading edge of cells at the wound front; α2β1-integrin expression was decreased in IFN-γ-treated cells compared with controls, whereas it was highest in IL-4- and IL-13-treated cells. These results demonstrate that IL-4 and IL-13 diminish the capacity of Calu-3 cells to maintain barrier function and repair wounds, whereas IFN-γ promotes epithelial restitution by enhancing barrier function and wound healing.
Isolated membrane vesicles as tools for analysis of epithelial transport.
Authors: Hopfer U
Published: December 2017

In recent years a methodology has been developed to use vesicles of the isolated brush border and basolateral plasma membranes for intestinal transport studies. The methodology and information gained with the vesicle systems are discussed using the examples of nonelectrolyte transport. In particular, results are presented on the mechanisms of D-glucose and neutral amino acid translocation across both plasma membranes and the coupling of Na+ with sugar and amino acid transport in the brush-border membrane. Furthermore, the kinetic parameters of the Na+-dependent glucose transport system, determined with an equilibrium exchange procedure, and the effects of semistarvation on the transport properties of the brush border membrane are described.
Dominant-subordinate relationships in castrated male mice bearing testosterone implants.
Authors: Maruniak J A , Desjardins C , Bronson F H
Published: December 2017

This study tested the hypothesis that subordination in male mice is directly related to reduced levels of circulating testosterone. Several measures of aggressiveness and submissiveness were quantified during or after daily encounters between pairs of intact males, castrated males, or castrated males bearing testosterone implants that were designed to maintain plasma testosterone titers at levels slightly above those of intact males. Behavioral measures included the total amount of fighting that resulted from daily pairing, the tendency for subordinates to fight back, and the relative frequency of urine marking. In all these regards, pairs of testosterone-implanted, castrated males formed dominant-subordinate relationships that involved apparently normal submissive behavior (compared to intact control pairs). Thus we found no evidence that submissive behavior was directly related to a reduction in circulating testosterone titers.
Secretory effects of secretin on isolated perfused porcine pancreas.
Authors: Jensen S L , Fahrenkrug J , Holst J J , Kühl C , Nielsen O V , Schaffalitzky de Muckadell O B
Published: December 2017

The effect of pure natural porcine secretin on endocrine and exocrine pancreatic secretion was studied in the totally isolated perfused porcine pancreas. The exocrine pancreatic responses to secretin correspond well with those obtained in the anesthetized pig. The lowest concentration of secretin observed to increase pancreatic secretion was 2.8 pmol/liter, whereas the maximum pancreatic responses were obtained at a secretin concentration of 92 pmol/liter. The infusion of secretin in concentrations ranging from 2.8 to 278 pmol/liter in the presence of a constant concentration of glucose (7.5, 5.0, or 3.5 mmol/liter) was without effect on the insulin and glucagon release. Infusion of secretin at a concentration of 834 pmol/liter in the presence of glucose at 7.5 mmol/liter provoked a significant (P less than 0.01) short-lived increase in insulin secretion. However, there was no effect on the glucagon secretion. The results of this study indicate that neither the augmented insulin response nor the suppression of glucagon elicited by oral glucose depend on secretin.
Hepatic transport kinetics and plasma disappearance curves: distributed modeling versus conventional approach.
Authors: Forker E L , Luxon B
Published: December 2017

The conventional approach to estimating hepatic transfer coefficients from the plasma disappearance curve ignores the effects of blood flow in the splanchnic and peripheral circulations. The effect of these simplifying assumptions on derived estimates of the rate constants has never been studied. To examine this problem we have constructed a distributed model that takes account of intrahepatic concentration profiles, nonuniform blood-flow distribution in the sinusoids, and delayed mixing in the peripheral circulation. Solutions to the new model constructed by numerical inversion of the Laplace transform afford a comparison between the new model and the conventional one. The sensitivity of the conventional equations to experimental error has also been evaluated. The results indicate that conventional estimates of the rate constants for hepatic uptake and cell-to-plasma efflux are subject to a systematic underestimate, the errors increasing rapidly with the initial extraction fraction. Estimates of the uptake constant obtained from the initial slope are especially susceptible to circulatory distortions and proved unacceptable even at low values of the initial extraction fraction. The liver content at 3 min did not in general provide a reliable index to these errors. In contrast to these problems, the conventional model returns generally accurate estimates of the steady-state plasma clearance and the rate constant for excretion.
Role of gastrin and insulin in postprandial disruption of migrating complex in dogs.
Authors: Eeckhout C , De Wever I , Peeters T , Hellemans J , Vantrappen G
Published: December 2017

The duration of the disruption of the interdigestive migrating myoelectric complex (MMC) by various test meals in dogs was correlated with changes in serum gastrin and insulin levels. The test meals consisted of milk protein, sucrose, arachis oil and medium chain triglycerides (MCT). Intravenous infusions of glucose 20% were also used. Electrical activity of the small intestine was registered by means of electrodes implanted over the entire length of the gut. Hormones were assayed by radioimmunoassay techniques. The insulin level rose significantly after both the glucose infusion and the sucrose meal. The rise was small after the milk protein meal and nothing after arachis oil and MCT. Gastrin level was not changed by arachis oil or MCT and rose slightly after sucrose and milk protein. The MMC was not disrupted by glucose infusions, but was disrupted for 5--7 h by archis oil and for 6--12 h by MCT. We conclude that in dogs neither gastrin nor insulin have an important role in the mechanism of disruption of the MMC after feeding.
Evidence for existence of cortical androgen-stimulating hormone.
Authors: Parker L N , Odell W D
Published: December 2017

An animal model using dexamethosone-suppressed, castrated dogs was developed to test the hypothesis that a pituitary hormone other than ACTH modulates adrenal androgen (AA) secretion. Plasma samples were obtained every 15 min during infusions of saline, synthetic alpha 1-24 corticotropin, porcine 1-39 corticotropin (ACTH), or bovine pituitary gland extract (PE) in a wide range of doses. Androstenedione (A), dehydroepiandrosterone (DHA), and cortisol (F) were quantified by radioimmunoassay. When the ratio of AA levels was related to those of F, in order to correct for ACTH content in the PE, the slopes of the dose-response curves for corticotropin and PE were different at the 0.01 level. For A the dose-response slope for the PE was 0.18 +/- 0.5 SE, whereas that of ACTH was 0.02 +/- 0.01. For the DHA response the slopes were 0.17 +/- 0.04 for the PE and 0.04 +/- 0.03 for ACTH. Related studies showed no increase in AA levels in response to luteinizing hormone-releasing hormone, bovine growth hormone (GH), bovine prolactin, ovine thyroid-stimulating hormone (TSH), or synthetic aqueous arginine vasopressin (AVP). We conclude that a pituitary factor other than ACTH, prolactin, GH, luteinizing hormone, follicle-stimulating hormone, TSH, or AVP may be responsible for the observed increase in AA concentrations.
Acid secretion by isolated primate gastric mucosa.
Authors: Rangachari P K
Published: December 2017

Pentagastrin, histamine, and acetylcholine stimulated acid secretion in the isolated rhesus (Macaca mulatta) gastric mucosa. The potencies were in the order pentagastrin greater than histamine greater than acetylcholine. However, histamine was more effective, the calculated maximal response being twice as great as for the other two agonists. Metiamide inhibited acid secretion induced by both histamine and pentagastrin. Whereas the inhibition of histamine by metiamide was surmountable, that of pentagastrin was not. Atropine inhibited the response to acetylcholine. Even in higher doses, atropine had no effect on histamine. With stimulation, transmucosal potential differences and resistances decreased; the changes were reversed on inhibition.
PGE1-induced intestinal secretion: mechanism of enhanced transmucosal protein efflux.
Authors: Granger D N , Shackleford J S , Taylor A E
Published: December 2017

The effects of local intra-arterial prostaglandin E1 (PGE1) infusion on net transmucosal volume and protein fluxes, lymphatic volume and protein fluxes, and regional hemodynamics were ascertained in autoperfused segments of cat ileum. After acquiring control values (and tissue samples) for the various parameters, PGE1 (5.0 microgram/min) was infused directly into the superior mesenteric artery. The PGE1 infusions resulted in dramatic increases in ileal lymphatic volume and protein fluxes and blood flow. Infusion of PGE1 caused a reversion of net mucosal volume absorption to net secretion and an increased loss of plasma proteins into the lumen. Ultrastructural analysis of tissue samples taken during the PGE1 infusion indicate major structural damage to the mucosal membrane. The physiological and ultrastructural data acquired in this study suggest that 1) the increased transmucosal protein efflux during intra-arterial PGE1 infusions results from an alteration in mucosal transcapillary fluid exchange and 2) a significant portion of PGE1-induced ileal secretion is passively mediated.
In vivo NMR evidence for moderate glucose accumulation in human skeletal muscle during hyperglycemia
Authors: Roussel R. , Velho G. , Carlier P. G. , Jouvensal L. , Bloch G.
Published: December 2017

Although the absence of intracellular (IC) free glucose is direct evidence of glucose transport being the rate-limiting step for muscle glucose disposal at euglycemia, the scarcity of data in humans during hyperglycemia precludes any definitive conclusion. In the present study, 13C and 31P in vivo nuclear magnetic resonance (NMR) data from two separate groups of subjects were combined to measure IC free glucose in the human skeletal muscle. When these noninvasive tools were used with an infusion of [1-13C]glucose, a steady-state concentration of 1.2 +/- 0.2 mmol IC glucose/l IC water was observed at the end of a 2-h hyperglycemic clamp with somatostatin infusion, during which glycemia was maintained at approximately 22 mmol/l and insulinemia at approximately 5 mU/l. Despite this moderate glucose accumulation, the persistence of a large transmembrane glucose gradient suggests that the posttransport steps do not play a significant role in the control of muscle glucose disposal in these specific conditions, relevant to insulinopenic diabetic patients.
Presence of renin within intramitochondrial dense bodies of the rat adrenal cortex
Authors: Peters J. , Kranzlin B. , Schaeffer S. , Zimmer J. , Resch S. , Bachmann S. , Gretz N. , Hackenthal E.
Published: December 2017

It has been suggested that tissue-specific expression of the genes of the renin-angiotensin system (RAS) leads to local generation of angiotensin (ANG) II with specific physiological implications. We demonstrate here that an intracellular RAS exists in adrenal glomerulosa cells; 60 h after bilateral nephrectomy and hemodialysis, renin and prorenin were eliminated from the circulation, whereas intra-adrenal renin content increased (control rats: 2 +/- 0.5 ng ANG I.mg-1.h-1; anephric rats: 25 +/- 2). Thus renin is produced locally within adrenal cells. We obtained immunocytochemical and biochemical evidence for the presence of renin within intramitochondrial dense bodies of the zona glomerulosa. After nephrectomy, dense bodies increased in number, size, and renin content (control rats: 2.5 +/- 0.7 ngANGI.mg-1.h-1; anephric rats: 43 +/- 7). Angiotensin-converting enzyme (ACE) was also present within mitochondria and their dense bodies. In addition, in adrenal cortex of anephric rats, giant dense bodies were observed, which contain renin and strongly react with an anti-angiotensinogen antibody. The localization of renin, ACE, and angiotensinogen at these sites provides new evidence for the existence of an intracellular adrenal RAS.
Mechanism of clearance and transfer of dipeptides by perfused human placenta
Authors: Adibi S. A. , Schenker S. , Morse E.
Published: December 2017

Glycylglutamine (Gly-Gln) is stable source of glutamine for parenteral nutrition. In the present study we have investigated whether this dipeptide is transferred intact across the human placenta. Although after 90 min of placental perfusion there was almost complete disappearance of Gly-Gln (100 microM) from the maternal compartment, only a small concentration of this dipeptide (< 6 microM) appeared in the fetal compartment. To investigate whether this transfer was due to transcellular transport, brush-border membrane vesicles of the human placenta were probed with [3H]Gly-Gln, which showed no uptake. To investigate whether hydrolysis was the mechanism of disappearance of Gly-Gln, the perfusion study was repeated with glycylsarcosine (Gly-Sar), which is resistant to hydrolysis. In sharp contrast to Gly-Gln, after 90 min of perfusion nearly 80% of Gly-Sar remained in the perfusate (half-life of 24 vs. 235 min). The rest of the Gly-Sar was recovered intact in the fetal compartment. The addition of Gly-Gln to the maternal compartment increased the accumulation of glycine, but not glutamine, in both the maternal and fetal compartments. In conclusion, our data suggest that 1) the mechanism of clearance of Gly-Gln by perfused human placenta is largely hydrolysis, whereas that of Gly-Sar is largely passive diffusion, and 2) the placenta has a greater preference for glutamine than for glycine.
Growth hormone upregulates gastrin and peptide YY gene expression
Authors: Gomez G. , Udupi V. , Qi X. , Lluis F. , Rajaraman S. , Thompson J. C. , Greeley G. H.
Published: December 2017

The purpose of these studies was to examine the effects of excess growth hormone (GH) on gastrin and peptide YY (PYY) gene expression. Transgenic mice with the bovine GH gene linked to a mouse metallothionein I promoter were used as a model of chronic GH excess. Antral gastrin mRNA and peptide levels were elevated significantly (P < 0.05) in GH transgenic mice compared with wild type littermates. Ileal PYY mRNA and ileal and colonic PYY levels were significantly elevated in GH transgenic mice compared with wild type littermates. The elevations in gastrin and PYY gene expression in GH transgenic mice were independent of food intake. Serum concentrations of gastrin and PYY were also elevated in GH transgenic mice. Immunohistochemical analysis showed that the density of PYY-containing cells in the colon of GH transgenic mice and wild type littermates did not differ. In addition, the mRNA and protein levels of chromogranin A, a marker of endocrine cells, were not increased in the colon of GH transgenic mice. Together, these data indicate that GH, insulin-like growth factor I, or both can upregulate gastrointestinal gastrin and PYY gene expression directly.
Effect of a glucokinase inhibitor on energy production and insulin release in pancreatic islets
Authors: Sweet I. R. , Li G. , Najafi H. , Berner D. , Matschinsky F. M.
Published: December 2017

Glucokinase has exclusively high control strength on glucose usage in the pancreatic beta-cell. However, glucokinase also has extraordinarily high control strength on insulin secretion, which is linked to the phosphate potential, [ATP]/([ADP][Pi]) (F.M. Matschinsky, Y.Liang, P. Kesavan, L. Wang, P. Froguel, G. Velho, D. Cohen, M.A. Permutt, Y. Tanizawa, T.L. Jetton, K. Niswender, and M.A. Magnuson. J. Clin. Invest. 92: 2092-2098, 1993). We propose that the ATP produced via the tricarboxylic acid cycle is approximately constant, irrespective of the glucose level. Furthermore, the component of ATP production that is derived from glycolysis and glycolytically derived NADH, which is shuttled into the mitochondria, is a critical signal controlling the ionic events leading to insulin secretion, as suggested previously (M. J. MacDonald. Diabetes 39: 1461-1466, 1990 and I.D. Dukes, M.S. McIntyre, R.J. Mertz, L.H. Philipson, M.W. Roe, B. Spencer, and J.F. Worley III. J. Biol. Chem. 269: 10979-10982, 1994). To test this hypothesis, glucose usage, oxidation, and insulin secretion were measured in cultured rat islets over a wide range of concentrations of glucose and mannoheptulose, an inhibitor of glucokinase. These data were fit to a mathematical model that predicts that glucokinase will govern the rate of glucose usage and ATP production and will also have a strong, but not complete, control over the rate of glucose oxidation, the phosphate potential, and insulin release. Mannoheptulose caused an inhibition of all three fluxes. The estimates of the mechanistic parameters of the model [maximal velocity (Vmax) and Michaelis constant for glucokinase, Vmax for hexokinase and glucose transport, and the inhibition constant of mannoheptulose to glucokinase] were similar to those obtained in vitro. Thus the data are consistent with a model in which the primary importance of glycolysis in transducing the glucose signal into changes of the phosphate potential imparts to glucokinase a high control strength on glucose-induced insulin secretion.
IGF-I and TGF-β1 have distinct effects on phenotype and proliferation of intestinal fibroblasts
Authors: Simmons James G. , Pucilowska Jolanta B. , Keku Temitope O. , Lund P. Kay
Published: December 2017

Insulin-like growth factor I (IGF-I) and transforming growth factor-β1 (TGF-β1) are upregulated in myofibroblasts at sites of fibrosis in experimental enterocolitis and in Crohn's disease (CD). We compared the sites of expression of IGF-I and TGF-β1 in a rat peptidoglycan-polysaccharide (PG-PS) model of chronic granulomatous enterocolitis and fibrosis. We used the human colonic CCD-18Co fibroblast/myofibroblast cell line to test the hypothesis that TGF-β1 and IGF-I interact to regulate proliferation, collagen synthesis, and activated phenotype typified by expression of α-smooth muscle actin and organization into stress fibers. IGF-I potently stimulated while TGF-β1 inhibited basal DNA synthesis. TGF-β1 and IGF-I each had similar but not additive effects to induce type I collagen. TGF-β1 but not IGF-I potently stimulated expression of α-smooth muscle actin and stress fiber formation. IGF-I in combination with TGF-β1 attenuated stress fiber formation without reducing α-smooth muscle actin expression. Stress fibers were not a prerequisite for increased collagen synthesis. TGF-β1 upregulated IGF-I mRNA, which led us to examine the effects of IGF-I in cells previously activated by TGF-β1 pretreatment. IGF-I potently stimulated proliferation of TGF-β1-activated myofibroblasts without reversing activated fibrogenic phenotype. We conclude that TGF-β1 and IGF-I both stimulate type I collagen synthesis but have differential effects on activated phenotype and proliferation. We propose that during intestinal inflammation, regulation of activated phenotype and proliferation may require sequential actions of TGF-β1 and IGF-I, but they may act in concert to increase collagen deposition.
Conservation of the Notch1 signaling pathway in gastrointestinal carcinoid cells
Authors: Kunnimalaiyaan Muthusamy , Traeger Kelly , Chen Herbert
Published: December 2017

Gastrointestinal (GI) carcinoid cells secrete multiple neuroendocrine (NE) markers and hormones including 5-hydroxytryptamine and chromogranin A. We were interested in determining whether activation of the Notch1 signal transduction pathway in carcinoid cells could modulate production of NE markers and hormones. Human pancreatic carcinoid cells (BON cells) were stably transduced with an estrogen-inducible Notch1 construct, creating BON-NIER cells. In the present study, we found that Notch1 is not detectable in human GI carcinoid tumor cells. The induction of Notch1 in human BON carcinoid cells led to high levels of functional Notch1, as measured by CBF-1 binding studies, resulting in activation of the Notch1 pathway. Similar to its developmental role in the GI tract, Notch1 pathway activation led to an increase in hairy enhancer of split 1 (HES-1) protein and a concomitant silencing of human Notch1/HES-1/achaete-scute homolog 1. Furthermore, Notch1 activation led to a significant reduction in NE markers. Most interestingly, activation of the Notch1 pathway caused a significant reduction in 5-hydroxytryptamine, an important bioactive hormone in carcinoid syndrome. In addition, persistent activation of the Notch1 pathway in BON cells led to a notable reduction in cellular proliferation. These results demonstrate that the Notch1 pathway, which plays a critical role in the differentiation of enteroendocrine cells, is highly conserved in the gut. Therefore, manipulation of the Notch1 signaling pathway may be useful for expanding the targets for therapeutic and palliative treatment of patients with carcinoid tumors.
The histone deacetylase inhibitor butyrate downregulates cyclin B1gene expression via a p21/WAF-1-dependent mechanism in human colon cancer cells
Authors: Archer Sonia Y. , Johnson Jennifer , Kim Hyun-Ju , Ma Qing , Mou Huizhong , Daesety Vishnuvardhan , Meng Shufen , Hodin Richard A.
Published: December 2017

Histone deacetylase (HDAC) inhibitors are showing promise as treatment for a variety of human cancers, but their precise mechanism of action has not been elucidated. We examined the effects of the HDAC inhibitor butyrate on colon cancer cells, focusing on its effect on the cell cycle promoter cyclin B1. In HT-29 cells, sodium butyrate-mediated growth inhibition is associated with a marked decrease in cyclin B1mRNA levels. The decrease in cyclin B1occurred in a delayed fashion (at 24 h), is completely blocked by concomitant treatment with protein synthesis inhibitors, and appears to be dependent on changes in transcription. Cyclin B1repression is linked to the differentiation process in colon cancer cells, not merely with growth arrest. The mechanism of cyclin B1repression by butyrate requires prolonged histone hyperacetylation and is at least partly dependent on p21 expression. In fact, p21/WAF-1 appears to directly repress a minimal cyclin B1promoter (−90 bp), a process that can be mediated by the amino-terminal portion of the p21 protein. These findings highlight key molecular mechanisms by which HDAC inhibitors mediate their beneficial effects on human cancer cells.
Chloroquine stimulates absorption and inhibits secretion of ileal water and electrolytes
Authors: Fogel R. , Sharp G. W. , Donowitz M.
Published: December 2017

The effects of chloroquine diphosphate, a drug with "'membrane-stabilizing" properties, were studied on basal ileal absorption and on ileal secretion induced by increased intracellular cAMP levels and calcium (serotonin). The studies were performed on rat (in vivo) and rabbit ileum (in vitro). Intraluminal chloroquine (10(-4) M) reversed cholera toxin- and theophylline-induced secretion in rat ileum but did not alter the cholera toxin- and theophylline-induced increases in cAMP content. Addition of chloroquine (10(-4) M) to the mucosal surface of rabbit ileum did not alter basal active electrolyte transport or the serotonin-induced decreased Na and Cl absorption but inhibited the theophylline-induced C1 secretion. Addition of chloroquine (10(-4)) M) to the serosal surface stimulated net Na and Cl absorption. This effect may involve intracellular calcium. Chloroquine increased the rabbit ileal calcium content and decreased 45Ca2+ influx from the serosal surface. Both the mucosal and serosal effects of chloroquine described led to a net increase in absorptive function of the intestine and should prove useful in developing treatment of diarrheal diseases.
Autoradiographic and kinetic demonstration of acinar heterogeneity of taurocholate transport
Authors: Groothuis G. M. , Hardonk M. J. , Keulemans K. P. , Nieuwenhuis P. , Meijer D. K.
Published: December 2017

A combination of autoradiography of water-soluble compounds and normal and retrograde perfusions was used to study whether there was a heterogeneity in bile salt transport between zone 1 and zone 3 hepatocytes of the rat and, if so, whether such a heterogeneity was due to the localization of these cells in the liver blood-stream, to intrinsic cellular differences, or to both. When a low dose of [3H]taurocholate was administered under pentobarbital sodium anesthesia in vivo or to normally perfused livers, the label was localized primarily in zone 1; in livers perfused in a retrograde direction, it appeared predominantly in zone 3. High doses of [3H]taurocholate administered in vivo and in normal and retrograde perfusions resulted in a more homogeneous labeling of the acini. The plasma disappearance of [3H]taurocholate was similar in normal and retrograde perfusions, but in the latter biliary excretion occurred at a considerably slower rate. From these results it is concluded that at low doses bile salts are primarily transported by zone 1 cells. Zone 3 cells appear to be able to take up taurocholate with ease, but their biliary excretion is slower compared with zone 1 cells.
Mucosal gastrin receptor. VIII. Sex-related differences in binding
Authors: Johnson L. R. , Peitsch W. , Takeuchi K.
Published: December 2017

The purpose of the current study was to determine whether gastrin binding differed between the sexes and, if so, to ascertain the factors responsible. Male and female rats were killed at various times from 15 to 60 days after birth. Binding capacities in male and female rats were equal until puberty. By day 40 there were significantly more receptors present in males than females. Females had 2.4 fmol receptor/mg prot by day 40, and this value remained constant into adulthood. In male rats the number of receptors continued to increase to 4.0 fmol/mg prot at day 60. Castration did not significantly decrease the number of receptors in male rats. Ovariectomy, however, increased the number of receptors in female rats to a value not significantly different from male controls. Ovariectomized females consumed more food and had higher serum gastrin levels. If ovariectomized rats were pair fed to the level of intact females, the number of receptors and serum gastrin levels decreased over those present in ovariectomized rats fed ad libitum but still remained significantly higher than those in intact females. Adrenalectomy of ovariectomized females caused a further increase in receptor number.
Electrical activity of longitudinal and circular muscle during peristalsis
Authors: Yokoyama S. , North R. A.
Published: December 2017

Action potentials were recorded simultaneously from the longitudinal and circular muscle layers of the guinea pig isolated small intestine. Both the graded reflex of the longitudinal muscle and the peristaltic reflex proper could be evoked by raising the intraluminal pressure. At low intraluminal pressures, intervals between spike bursts of the circular muscle were longer than those of the longitudinal muscle. The higher the intraluminal pressure, the shorter became the intervals between spike bursts in the circular muscle, until both muscle layers showed synchronous discharge of action potentials. Tetrodotoxin (100 nM) abolished the excitation of both circular and longitudinal muscles produced by raising intraluminal pressure. Hexamethonium (280 microM) abolished excitation of the circular muscle but not that of the longitudinal muscle. Atropine (100 nM) reduced the excitatory effects of raising pressure on both muscle layers but did not abolish them. The atropine-resistant excitation of the circular, but not the longitudinal, muscle was reversibly blocked by exposure to substance P (100–500 nM). Chymotrypsin (200 micrograms/ml) reversibly abolished the atropine-resistant excitation of the circular muscle. It was concluded that during peristalsis both longitudinal and circular muscle layers are activated synchronously; muscle activation during peristalsis is not entirely cholinergic but may involve in addition a substance P-like peptide.
Neural control of canine small intestinal interdigestive myoelectric complexes
Authors: Heppell J. , Kelly K. A. , Sarr M. G.
Published: December 2017

Our aim was to determine the role of the extrinsic and intrinsic nerves in the regulation of the small intestinal interdigestive myoelectric complex (IMC). In five dogs, the extrinsic nerves of the jejunoileum were divided, but the bowel was left in situ with its wall intact. After recovery, IMCs were detected in the duodenum (mean IMC period +/- SE = 188 +/- 19 min), from where most migrated distally into and through the jejunum (84%). In addition, extra IMCs appeared in the jejunum. Thus, the mean period of the IMCs in the jejunum (134 +/- 10 min) was shorter than in the duodenum (P less than 0.05). In contrast, after enteric transection and reanastomosis at the ligament of Treitz and at a site 75 cm distal to the ligament, fewer duodenal IMCs migrated into the jejunum (only 61%). However, extra IMCs still arose in the jejunum, so that the mean period of IMCs in the jejunum (111 +/- 5 min) was now similar to that in the duodenum (128 +/- 4 min, P greater than 0.05). Our data were consistent with the hypothesis that extrinsic nerves regulate the frequency of the small intestinal IMCs, while an intact enteric wall aids in their distal propagation.
Effects of trifluoperazine, ouabain, and ethacrynic acid on intestinal calcium transport
Authors: Favus M. J. , Angeid-Backman E. , Breyer M. D. , Coe F. L.
Published: December 2017

Bidirectional steady-state calcium fluxes were measured in vitro under short-circuited conditions in segments of rat duodenum and descending colon. The calcium-activated ATPase (Ca-ATPase) inhibitor trifluoperazine (TFP, 0.1 mM) reduced net calcium absorption in both tissues by decreasing the absorptive flux from mucosa to serosa (Jm leads to s) without consistently altering the secretory flux from serosa to mucosa. 1,25-Dihydroxyvitamin D3 administration (50 ng/day for 4 days) increased net calcium absorption by increasing Jm leads to s, and TFP reduced Jm leads to s to the same extent across tissues from vehicle- or 1,25-dihydroxyvitamin D3-treated animals. Na-K-ATPase inhibitors ouabain and ethacrynic acid both reduced short-circuit current without affecting calcium fluxes. These data suggest that Ca-ATPase, located in the basolateral membrane of intestinal epithelial cells, plays a role in the transepithelial transport of calcium. More general effects of TFP on intestinal epithelium may also contribute to the reduction in calcium fluxes. Duodenal and descending colon calcium transport appears independent of transcellular sodium transport mediated by Na-K-ATPase.