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Factors controlling cardiac sympathetic nerve activity (CSNA) in the normal state and those causing the large increase in activity in heart failure (HF) remain unclear. We hypothesized from previous clinical findings that activation of cardiac mechanoreceptors by the increased blood volume in HF may stimulate sympathetic nerve activity (SNA), particularly to the heart via cardiocardiac reflexes. To investigate the effect of volume expansion and depletion on CSNA we have made multiunit recordings of CSNA in conscious normal sheep and sheep paced into HF. In HF sheep (n = 9) compared with normal sheep (n = 9), resting levels of CSNA were significantly higher (34 [+ or -] 5 vs. 93 [+ or -] 2 bursts/100 heart beats, P < 0.05), mean arterial pressure was lower (76 [+ or -] 3 vs. 87 [+ or -] 2 mmHg; P < 0.05), and central venous pressure (CVP) was greater (3.0 [+ or -] 1.0 vs. 0.0 [+ or -] 1.0 mmHg; P < 0.05). In normal sheep (n = 6), hemorrhage (400 ml over 30 min) was associated with a significant increase in CSNA (179 [+ or -] 16%) with a decrease in CVP (2.7 [+ or -] 0.7 mmHg). Volume expansion (400 ml Gelofusine over 30 min) significantly decreased CSNA (35 [+ or -] 12%) and increased CVP (4.7 [+ or -] 1.0 mmHg). In HF sheep (n = 6) the responses of CSNA to both volume expansion and hemorrhage were severely blunted with no significant changes in CSNA or heart rate with either stimulus. In summary, these studies in a large conscious mammal demonstrate that in the normal state directly recorded CSNA increased with volume depletion and decreased with volume loading. In contrast, both of these responses were severely blunted in HF with no significant changes in CSNA during either hemorrhage or volume expansion. heart failure; sympathetic nervous system; hemorrhage; cardiac volume

The publication of a research paper in a journal completes the research activity and an unpublished research work is considered as incomplete in modern science activities. This trend of publishing originated in the seventeenth century. Henry Oldenberg in 1665 based on the ideology that science is a cooperative activity and 'growth of knowledge is incremental' , published in the first journal 'philosophical transactions' . The publication of research works in the form of small articles enable the development of science, as it enables other researchers to modify an experiment performed earlier and published in a journal.

The evolution of Forum for editing and peer-reviewing of articles that use mathematical modeling in biological science is the response of American Physiological Society to inadequate attention received by modeling papers in the past. The pioneering standards advocated by Gene Yates for authors and editors are further developed in the areas of sensitivity analysis and uniform nomenclature. Finding competent editors and reviewers and editorial delays are difficulties that need to be overcome.

The use of the fluorescent chloride-indicator dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) in studies of concomitant transmembrane fluxes of Cl- and H+ was investigated. The results showed that the fluorescence intensity of SPQ decreased with increasing Cl- concentration. However, it was discovered that SPQ fluorescence intensity also progressively decreased as pH was decreased from 7 to 3, independently of the presence of either Cl- or gluconate. Further study showed that the common buffers HEPES and MES, previously thought as inert, quenched SPQ fluorescence in a pH-dependent manner.

This paper proposes a new, two-dimensional convection-diffusion model for macromolecular transport in heart valves based on horseradish peroxidase (HRP) experiments on rats presented in the first of the papers in this series (Part I; Zeng Z, Yin Y, Huang AL, Jan KM, Rumschitzki DS. Am J Physiol Heart Circ Physiol 292: H2664-H2670, 2007). Experiments require two valvular intimae, one underneath each endothelium. Tompkins et al. (Tompkins RG, Schnitzer JJ, Yarmush ML. Circ Res 64: 1213-1223, 1989) found large variations in shape and magnitude in transvalvular [sup.125]I-labeled low-density lipoprotein (LDL) profiles from identical experiments on four squirrel monkeys. Their one-dimensional, uniform-medium diffusion-only model fit three parameters independently for each profile; data variability resulted in large parameter spreads. Our theory aims to explain their data with one parameter set. It uses measured parameters and some aortic values but fits the endothelial mass transfer coefficient ([k.sub.a] = [k.sub.v] = 1.63 x [10.sup.-8] cm/s, where subscripts a and v indicate aortic aspect and ventricular aspect, respectively) and middle layer permeability ([K.sub.p2] = 2.28 x [10.sup.-16] [cm.sup.2]) and LDL diffusion coefficient [[D.sub.2](LDL) = 5.93 x [10.sup.-9] [cm.sup.2]/s], using one of Tompkins et al.'s profiles, and fixes them throughout. It accurately predicts Part I's rapid localized HRP leakage spot growth rate in rat leaflets that results from the intima's much sparser structure, dictating its far larger transport parameters [[K.sub.p1] = 1.10 x [10.sup.-12] [cm.sup.2], [D.sub.1] (LDL/HRP) 102/409 x [10.sup.-7] [cm.sup.2]/s] than the middle layer. This contrasts with large arteries with similarly large HRP spots, since the valve has no internal elastic lamina. The model quantitatively explains all of Tompkins et al.'s monkey profiles with these same parameters. Different numbers and locations of isolated macromolecular leaks on both aspects and different section-leak(s) distances yield all profiles. convection-diffusion; two-dimensional transport; variation in LDL profiles doi:10.1152/ajpheart.00608.2006

CTP:phosphocholine cytidylyltransferase inhibition by ceramide via PKC-[Alpha], p38 MAPK, [cPLA.sub.2], and 5-lipoxygenase. Am J Physiol Lung Cell Mol Physiol 281: L108-L118, 2001.--In a companion paper (Vivekananda J, Smith D, and King RJ. Am J Physiol Lung Cell Mol Physiol 281: L98-L107, 2001), we demonstrated that tumor necrosis factor (TNF)-[Alpha] inhibited the activity of CTP:phosphocholine cytidylyltransferase (CT), the rate-limiting enzyme in the de novo synthesis of phosphatidylcholine (PC), and that its actions were likely exerted through a metabolite of sphingomyelin. In this paper, we explore the signaling pathway employed by TNF-[Alpha] using [C.sub.2] ceramide as a cell-penetrating sphingolipid representative of the metabolites induced by TNF-[Alpha]. We found that in H441 cells, as reported in other cell types, cytosolic phospholipase [A.sub.2] ([cPLA.sub.2]) is activated by TNF-[Alpha]. We also observed that the inhibiting action of [C.sub.2] ceramide on CT requires protein kinase C-[Alpha], p38 mitogen-activated protein kinase, and [cPLA.sub.2]. The actions of [C.sub.2] ceramide on CT activity can be duplicated by adding 2 [micro]M lysoPC to these cells. Furthermore, we found that the effects of [C.sub.2] ceramide are dependent on 5-lipoxygenase but that cyclooxygenase II is unimportant. We hypothesize that CT activity is inhibited by the lysoPC generated as a consequence of the activation of [cPLA.sub.2] by protein kinase C-[Alpha] and p38 mitogen-activated protein kinase. The other product of the activation of [cPLA.sub.2], arachidonic acid, is a substrate for the synthesis of leukotrienes, which raise intracellular [Ca.sup.2+] levels and complete the activation of [cPLA.sub.2]. lung injury; pulmonary surfactant; phosphatidylcholine synthesis; leukotrienes; cytidine 5'-triphosphate; protein kinase C; mitogen-activated protein kinase; cytosolic phospholipase [A.sub.2]

Gyenge, C. C., B. D. Bowen, R. K. Reed, and J. L. Bert. Transport of fluid and solutes in the body. I. Formulation of a mathematical model. Am. J. Physiol. 277 (Heart Circ. Physiol. 46): H1215-H1227, 1999.--A compartmental model of short-term whole body fluid, protein, and ion distribution and transport is formulated. The model comprises four compartments: a vascular and an interstitial compartment, each with an embedded cellular compartment. The present paper discusses the assumptions on which the model is based and describes the equations that make up the model. Fluid and protein transport parameters from a previously validated model as well as ionic exchange parameters from the literature or from statistical estimation [see companion paper: C. C. Gyenge, B. D. Bowen, R. K. Reed, and J. L. Bert. Am. J. Physiol. 277 (Heart Circ. Physiol. 46): H1228-H1240, 1999] are used in formulating the model. The dynamic model has the ability to simulate 1) transport across the capillary membrane of fluid, proteins, and small ions and their distribution between the vascular and interstitial compartments; 2) the changes in extracellular osmolarity; 3) the distribution and transport of water and ions associated with each of the cellular compartments; 4) the cellular transmembrane potential; and 5) the changes of volume in the four fluid compartments. The validation and testing of the proposed model against available experimental data are presented in the companion paper. hyperosmolarity; cell volume; interstitial volume; plasma volume expansion; plasma osmolarity

Myocardial infarction has been the leading cause of morbidity and mortality in developed countries over the past few decades. The transplantation of cardiomyocytes offers a potential method of treatment. However, cardiomyocytes are in high demand and their supply is extremely limited. Embryonic stem cells (ESCs), which have been isolated from the inner cell mass of blastocysts, can self-renew and are pluripotent, meaning they have the ability to develop into any type of cell, including cardiomyocytes. This suggests that ESCs could be a good source of genuine cardiomyocytes for future therapeutic purposes. However, problems with the yield and purity of ESC-derived cardiomyocytes, among other hurdles for the therapeutic application of ESC-derived cardiomyocytes (e.g., potential immunorejection and tumor formation problems), need to be overcome before these cells can be used effectively for cell replacement therapy. ESC-derived cardiomyocytes consist of nodal, atrial, and ventricular cardiomyocytes. Specifically, for treatment of myocardial infarction, transplantation of a sufficient quantity of ventricular cardiomyocytes, rather than nodal or atrial cardiomyocytes, is preferred. Hence, it is important to find ways of increasing the yield and purity of specific types of cardiomyocytes. Atrial and ventricular cardiomyocytes have differential expression of genes (transcription factors, structural proteins, ion channels, etc.) and are functionally distinct. This paper presents a thorough review of differential gene expression in atrial and ventricular myocytes, their expression throughout development, and their regulation. An understanding of the molecular and functional differences between atrial and ventricular myocytes allows discussion of potential strategies for preferentially directing ESCs to differentiate into chamber-specific cells, or for fine tuning the ESC-derived cardiomyocytes into specific electrical and contractile phenotypes resembling chamber-specific cells. atrial cardiomyocytes; ventricular cardiomyocytes; gene regulations; therapeutic application doi: 10.1152/ajpcell.00402.2009.

Extracellular matrix (ECM) remodeling occurs during normal homeostasis and also plays an important role during development, tissue repair, and in various disease processes. ECM remodeling involves changes in the synthesis, deposition, and degradation of ECM molecules. ECM molecules can be degraded extracellularly, as well as intracellularly following endocytosis. Our data show that the ECM protein fibronectin is an important regulator of ECM remodeling. We previously showed that agents that inhibit the polymerization of fibronectin into ECM fibrils promote the loss of preexisting fibronectin matrix and accelerate fibronectin endocytosis and degradation. In this paper we show that inhibition of fibronectin polymerization leads to the loss of collagen I matrix fibrils and a corresponding increase in the levels of endocytosed collagen I. In contrast, manipulations that stabilize fibronectin matrix fibrils, such as caveolin-1 depletion, stabilize collagen I matrix fibrils and cause a decrease in ECM collagen I endocytosis. Our data also show that endocytosis of ECM collagen I is regulated by both [beta]1 integrins and Endo180/urokinase plasminogen activator associated protein (uPARAP). Unexpectedly, Endo180/uPARAP was also shown to promote the endocytosis of fibronectin from the ECM. These data demonstrate that fibronectin polymerization regulates the remodeling of ECM collagen I, in part, by regulating collagen I endocytosis. Furthermore, these data show that processes that regulate ECM deposition coordinately regulate the removal of proteins from the ECM. These data highlight the complexity of ECM remodeling. This multifaceted regulatory process may be important to ensure tight regulation of ECM fibronectin and collagen I levels. extracellular matrix; integrin; endocytosis; Endo180; urokinase plasminogen activator-associated protein doi: 10.1152/ajpcell.00341.2009.

The double knockout mouse for utrophin and dystrophin ([utr.sup.-/-]/mdx) has been proposed to be a better model of Duchenne Muscular Dystrophy (DMD) than the mdx mouse because the former displays more similar muscle pathology to that of the DMD patients. In this paper the properties of action potentials (APs) and [Ca.sup.2+] transients elicited by single and repetitive stimulation were studied to understand the excitation-contraction (EC) coupling alterations observed in muscle fibers from mdx and [utr.sup.-/-]/mdx mice. Based on the comparison of the AP durations with those of fibers from wild-type (WT) mice, fibers from both mdx and [utr.sup.-/-]/mdx mice could be divided in two groups: fibers with WT-like APs (group 1) and fibers with significantly longer APs (group 2). Although the proportion of fibers in group 2 was larger in [utr.sup.-/-]/mdx (36%) than in mdx mice (27%), the [Ca.sup.2+] release elicited by single stimulation was found to be similarly depressed (32-38%) in [utr.sup.-/-]/mdx and mdx fibers compared with WT counterparts regardless of the fiber's group. Stimulation at 100 Hz revealed that, with the exception of those from [utr.sup.-/-]/mdx mice, group 1 fibers were able to sustain [Ca.sup.2+] release for longer than group 2 fibers, which displayed an abrupt limitation even at the onset of the train. The differences in behavior between fibers in groups 1 and 2 became almost unnoticeable at 50 Hz stimulation. In general, fibers from [utr.sup.-/-]/mdx mice seem to display more persistent alterations in the EC coupling than those observed in the mdx model. muscular dystrophy; mammalian skeletal muscle; fluorescence microscopy doi: 10.1152/ajpcell.00428.2009.

Am J Physiol Endocrinol Metab 298: E1-E7, 2010. First published November 3, 2009; doi: 10.1152/ajpendo.00562.2009.--Autophagy is a major degradative pathway(s) by which intracellular components are delivered into the lysosomes. It is largely implicated in determining cell death and survival because it eliminates unnecessary, damaged, and/or potentially harmful cellular products and organelles and is an important source for nutrients and energy production under conditions of external nutrient deficiency. As such, autophagy has been suggested to contribute to the regulation of carbohydrate and protein metabolism during fasting. Recently, three papers implicated a role for autophagy in cellular lipid metabolism as well. This Perspectives article presents these novel findings in the context of prior studies on the role of autophagy and lysosomes in metabolic and energy regulation, discusses their points of agreement and opposing propositions, and outlines key outstanding questions. lipolysis; liquid droplet biogenesis and breakdown; liquid droplet-associated proteins; lysosomal proteolysis

Am J Physiol Cell Physiol 298: C124-C131, 2010. First published October 28, 2009; doi:10.1152/ajpcell.00220.2009.--SMCT1 is a Na-coupled cotransporter of short chain monocarboxylates, which is expressed in the apical membrane of diverse epithelia such as colon, renal cortex, and thyroid. We previously reported that SMCT1 cotransport was reduced by extracellular [Cl.sup.-] replacement with [cyclamate.sup.-] and that the protein exhibited an ostensible anionic leak current. In this paper, we have revisited the interaction between small monovalent anions and SMCT cotransport and leak currents. We found that the apparent [Cl.sup.-] dependence of cotransport was due to inhibition of this protein by the replacement anion cyclamate, whereas several other replacement anions function as substrates for SMCT1; a suitable replacement anion ([MES.sup.-]) was identified. The observed outward leak currents represented anionic influx and favored larger anions (N[O.sub.3.sup.-]>[I.sup.-]>[Br.sup.-]>[Cl.sup.-]); currents in excess of 1 [micro]A (at +50 mV) could be observed and exhibited a quasilinear relationship with anion concentrations up to 100 mM. Application of 25 mM bicarbonate did not produce measurable leak currents. The leak current displayed outward rectification, which disappeared when external [Na.sup.+] was replaced by N-methyl-D-[glucamine.sup.+]. More precisely, external [Na.sup.+] blocked the leak current in both directions, but its [K.sub.i] value rose rapidly when membrane potential became positive. Thus SMCT1 possesses a anionic leak current that becomes significant whenever external [Na.sup.+] concentration is reduced. The presence of this leak current may represent a second function for SMCT1 in addition to cotransporting short chain fatty acids, and future experiments will determine whether this function serves a physiological role in tissues where SMCT1 is expressed. iodide; cyclamate

Ares-Carrasco S, Picatoste B, Benito-Martin A, Zubiri I, Sanz AB, Sanchez-Nino MD, Ortiz A, Egido J, Tunon J, Lorenzo O. Myocardial fibrosis and apoptosis, but not inflammation, are present in long-term experimental diabetes. Am J Physiol Heart Circ Physiol 297: H2109-H2119, 2009. First published October 9, 2009; doi: 10.1152/ajpheart.00157.2009.--The aim of this paper is to study the myocardial damage secondary to long-term streptozotocin-induced type 1 diabetes mellitus (DM 1). Normotensive and spontaneously hypertensive rats (SHR) received either streptozotocin injections or vehicle. After 22 or 6 wk, DM1, SHR, DM1/SHR, and control rats were killed, and the left ventricles studied by histology, quantitative PCR, Western blot, ELISA, and electromobility shift assay. Cardiomyocyte cultures were also performed. The expression of profibrotic factors, transforming growth factor-[beta] (TGF-[[beta].sub.1]), connective tissue growth factor, and matrix proteins was increased, and the TGF-[[beta].sub.1]-linked transcription factors phospho-Smad3/4 and activator protein-1 were activated in the DM1 myocardium. Proapoptotic molecules FasL, Fas, Bax, and cleaved caspase-3 were also augmented. Myocardial injury in long-term hypertension shared these features. In addition, hypertension was associated with activation of NF-[kappa]B, increased inflammatory cell infiltrate, and expression of the mediators [interleukin-1[beta] (IL-[beta]), tumor necrosis factor-[alpha], monocyte chemoattractant protein 1, vascular cell adhesion molecule 1, angiotensinogen, and oxidants], which were absent in long-term DM1. At this stage, the combination of DM1 and hypertension resulted in nonsignificant additive effects. Moreover, the coexistence of DM1 blunted the inflammatory response to hypertension. Anti-inflammatory IL-10 and antioxidants were induced in long-term DM1 and DM1/SHR hearts. Myocardial inflammation was, however, observed in the short-term model. In cultured cardiomyocytes, IL-10, TGF-[[beta].sub.1], and catalase blocked the glucose-stimulated expression of proinflammatory genes. Fibrosis and apoptosis are features of long-term myocardial damage in experimental DM1. Associated hypertension does not induce additional changes. Myocardial inflammation is present in hypertension and short-term DM1, but is not a key feature in long-term DM 1. Local reduction of proinflammatory factors and expression of anti-inflammatory and antioxidant molecules may underlie this effect. hypertension; inflammation; heart doi: 10.1152/ajpheart.00157.2009

Excessive inflammation in cystic fibrosis (CF) lung disease is a contributor to progressive pulmonary decline. Effective and well-tolerated anti-inflammatory therapy may preserve lung function, thereby improving quality and length of life. In this paper, we assess the anti-inflammatory effects of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-l,9(11)-dien-28-oic acid (CDDO) in preclinical models of CF airway inflammation. In our experiments, mice carrying the R117H Cftr mutation have significantly reduced airway inflammatory responses to both LPS and flagellin when treated with CDDO before inflammatory challenge. Anti-inflammatory effects observed include reduced airway neutrophilia, reduced concentrations of proinflammatory cytokines and chemokines, and reduced weight loss. Our findings with the synthetic triterpenoids in multiple cell culture models of CF human airway epithelia agree with effects previously described in other disease models (e.g., neoplastic cells). These include the ability to reduce NF-[kappa]B activation while increasing nuclear factor erythroid-related factor 2 (Nrf2) activity. As these two signaling pathways appear to be pivotal in regulating the net inflammatory response in the CF airway, these compounds are a promising potential anti-inflammatory therapy for CF lung disease. anti-inflammatory therapy; antioxidant; nuclear factor-[kappa]B; nuclear factor erythroid-related factor 2; synthetic triterpenoid doi: 10.1152/ajplung.00171.2009.

Using adapted retroviruses for gene delivery is a modern and powerful tool in biological research as well as a promising approach for gene therapy. An important limitation for the extensive use of retroviral vectors is the low infection rate in target cells such as pulmonary vascular endothelial cells due to the insufficient infectivity of standard retrovirus supernatants that can only be overcome by complicated methods of vires concentration. This paper describes two easy methods to augment target cell infectivity, first by increasing the retroviral titer in the medium collected from packaging cells by stimulation of viral propagation with dexamethasone, and second, by increasing target cell sensitivity to retroviral infection by the glucocorticoid receptor antagonist, mifepristone. Using this method, we increased the infectivity of pulmonary microvascular endothelial cells from 16% to 85%. We demonstrate that mifepristone increased the susceptibility of target cells to retroviruses without increasing the viral titer of the supernatant. Dexamethasone, but not mifepristone, increased expression of delivered proteins such as GFP that are important for early identification of infected cells. Each, or both step(s), may be included in a standard protocol for retrovirus propagation and infection of target cells. retroviruses; pulmonary artery endothelium; gene delivery

In 1963, Lancet published a paper by Randle et al. that proposed a 'glucose-fatty acid cycle' to describe fuel flux between and fuel selection by tissues. The original biochemical mechanism explained the inhibition of glucose oxidation by fatty acids. Since then, the principle has been confirmed by many investigators. At the same time, many new mechanisms controlling the utilization of glucose and fatty acids have been discovered. Here, we review the known short- and long-term mechanisms involved in the control of glucose and fatty acid utilization at the cytoplasmic and mitochondrial level in mammalian muscle and liver under normal and pathophysiological conditions. They include allosteric control, reversible phosphorylation, and the expression of key enzymes. However, the complexity is formidable. We suggest that not all chapters of the Randle cycle have been written. adenosine 5'-monophosphate-activated protein kinase; fatty acids; glucose; heart; insulin resistance; liver; mitochondria; peroxisome proliferator-activated receptor; skeletal muscle; type 2 diabetes

Vitamin E is a generic term used to indicate all tocopherol (TOC) and tocotrienol (TT) derivates. In the last few years, several papers have shown that a TT-rich fraction (TTRF) extracted from palm oil inhibits proliferation and induces apoptosis in a large number of cancer cells. However, the molecular mechanism(s) involved in TT action is still unclear. In the present study, we proposed for the first time a novel mechanism for TT activity that involves estrogen receptor (ER) signaling. In silico simulations and in vitro binding analyses indicated a high affinity of TTs for ER[beta] but not for ER[alpha]. In addition, in ER[beta]-containing MDA-MB-231 breast cancer cells, we demonstrated that TTs increase the ER[beta] translocation into the nucleus, which in turn activates estrogen-responsive genes (MIC-1, EGR-1 and cathepsin D), as demonstrated by cell preincubation with the ER inhibitor ICI-182,780. Finally, we observed that TT treatment is associated with alteration of cell morphology, DNA fragmentation, and caspase-3 activation. Altogether, these experiments elucidated the molecular mechanism underling [gamma]- and [delta]-TT effects. estrogen receptor-[beta]; breast cancer; apoptosis; tocopherol; nuclear receptor

Mutations in the ryanodine receptor (RyR) have been linked to exercise-induced sudden cardiac death. However, the precise sequence of events linking RyR channel mutations to a whole heart arrhythmia is not completely understood. In this paper, we apply a detailed, mathematical model of subcellular calcium (Ca) release, coupled to membrane voltage, to study how defective RyR channels can induce arrhythmogenic-triggered activity. In particular, we show that subcellular Ca activity, such as spontaneous Ca sparks and Ca waves, is highly sensitive to coupled gating between RyR channels in clusters. We show that small changes in coupled gating can induce aberrant Ca release activity, which, under Ca overload conditions, can induce delayed afterdepolarization (DAD). We systematically investigate the properties of subcellular Ca during DAD induction and show that the voltage time course during a DAD is dependent on the timing and number of spontaneous Ca sparks that transition to Ca waves. These results provide a detailed mechanism for the role of coupled gating in the genesis of triggered arrhythmias. calcium; modeling; arrhythmia

In this paper, we describe our design for a new electrohydraulic (EH) pump-driven renal perfusion pressure (RPP)-regulatory system capable of implementing precise and rapid RPP regulation in experimental animals. Without this automated system, RPP is manually controlled via a blood pressure clamp, and the imprecision in this method leads to compromised RPP data. This motivated us to develop an EH pump-driven closed-loop blood pressure regulatory system based on flow-mediated occlusion using the vascular occlusive cuff technique. A closed-loop servo-controller system based on a proportional plus integral (PI) controller was designed using the dynamic feedback RPP signal from animals. In vivo performance was evaluated via flow-mediated RPP occlusion, maintenance, and release responses during baseline and ANG II-infused conditions. A step change of -30 mmHg, referenced to normal RPP, was applied to Sprague-Dawley rats with the proposed system to assess the performance of the PI controller. The PI's performance was compared against manual control of blood pressure clamp to regulate RPP. Rapid RPP occlusion (within 3 s) and a release time of ~0.3 s were obtained for the PI controller for both baseline and ANG II infusion conditions, in which the former condition was significantly better than manual control. We concluded that the proposed EH RPP-regulatory system could fulfill in vivo needs to study various pressure-flow relationships in diverse fields of physiology, in particular, studying the dynamics of the renal autoregulatory mechanisms. renal perfusion pressure; proportional plus integral compensation; tubuloglomerular feedback; myogenic

Endogenous insulin-like growth factor-I (IGF-I) regulates intestinal smooth muscle growth by concomitantly stimulating proliferation and inhibiting apoptosis. IGFI-stimulated growth is augmented by the [[alpha].sub.v][[beta].sub.3] integrin ligands vitronectin and fibronectin. IGF-I expression in smooth muscle is increased in both TNBS-induced colitis and Crohn's disease. We hypothesized that intestinal inflammation increased vitronectin and fibronectin expression by smooth muscle and, along with IGF-I upregulation, increased intestinal muscle growth. Intestinal smooth muscle cells were examined 7 days following the induction of TNBS-induced colitis. Although [[alpha].sub.v][[beta].sub.3] integrin expression was not altered by TNBS-induced colitis, vitronectin and fibronectin levels were increased by 80 [+ or -] 10% and 90 [+ or -] 15%, above control levels, respectively. Basal IGF-I receptor phosphorylation in inflamed muscle from TNBS-treated rats was increased by 86 [+ or -] 8% over vehicle-treated controls. Basal ERK1/2, p70S6 kinase, and GSK-3[beta] phosphorylation in muscle cells of TNBS-treated rats were also increased by 140-180%. TNBS treatment increased basal muscle cell proliferation by 130 [+ or -] 15% and decreased apoptosis by 20 [+ or -] 2% compared with that in vehicle-treated controls. The changes in proliferation and apoptosis were reversed by an IGF-I receptor tyrosine kinase inhibitor or an [[alpha].sub.v][[beta].sub.3] integrin antagonist. The results suggest that smooth muscle hyperplasia in TNBS-induced colitis partly results from the upregulation of endogenous IGF-I and ligands of [[alpha].sub.v][[beta].sub.3] integrin that mediate increased smooth muscle cell proliferation and decreased apoptosis. This paper has identified one mechanism regulating smooth muscle hyperplasia, a feature of stricture formation that occurs in the chronically inflamed intestine of TNBS-induced colitis and potentially Crohn's disease. Crohn's disease; insulin-like growth factor

Vascular supply is an obvious requirement for all organs. In addition to oxygen and nutrients, blood flow also transports essential trace elements. Iodine, which is a key element in thyroid hormone synthesis, is one of them. An inverse relationship exists between the expansion of the thyroid microvasculature and the local availability of iodine. This microvascular trace element-dependent regulation is unique and contributes to keep steady the iodide delivery to the thyroid. Signals involved in this regulation, such as VEGF-A, originate from thyrocytes as early TSH-independent responses to iodide scarcity. The question raised in this paper is how thyrocytes, facing an acute drop in intracellular stores of iodine, generate angiogenic signals acting on adjacent capillaries. Using in vitro models of rat and human thyroid cells, we show for the first time that the deficit in iodine is related to the release of VEGF-A via a reactive oxygen species/hypoxia-inducible factor-1-dependent pathway. thyroid function; angiogenesis; vascular endothelial growth factor A; oxidative stress; hypoxia-inducible factor; reactive oxygen species

The inflammatory bowel diseases (Crohn' s disease; ulcerative colitis) are idiopathic chronic inflammatory disorders of the intestine and/or colon. A major advancement in our understanding of the pathogenesis of these diseases has been the development of mouse models of chronic gut inflammation. One model that has been instrumental in delineating the immunological mechanisms responsible for the induction as well as regulation of intestinal inflammation is the T cell transfer model of chronic colitis. This paper presents a detailed protocol describing the methods used to induce chronic colitis in mice. Special attention is given to the immunological concepts that explain disease pathogenesis in this model, considerations and potential pitfalls in using this model, and finally different 'tricks' that we have learned over the past 12 years that have allowed us to develop a more simplified version of this model of experimental IBD. adaptive immune system; T cell trafficking; inflammation; inflammatory bowel disease; cytokines; IL-10-deficient mice

The inward L-dihydroxyphenylalanine (L-dopa) transport supplies renal proximal tubule cells (PTCs) with the precursor for dopamine synthesis. We have previously described insulin-induced stimulation of L-dopa uptake into PTCs. In the present paper we examined insulin-related signaling pathways involved in the increase of L-dopa transport into isolated rat PTCs. Insulin (50-500 [micro]U/ml) increased L-dopa uptake by PTCs, reaching the maximal increment (60% over the control) at 200 [micro]U/ml. At this concentration, insulin also increased insulin receptor tyrosine phosphorylation. Both effects were abrogated by the tyrosine kinase inhibitor genistein (5 [micro]M). In line, inhibition of the protein tyrosine phosphatase by pervanadate (0.2-100 [micro]M) caused a concentration-dependent increase in both the uptake of L-dopa (up to 400%) and protein tyrosine phosphorylation. A synergistic effect between pervanadate and insulin on L-dopa uptake was observed only when threshold (0.2 [micro]M), but not maximal (5 [micro]M), concentrations of pervanadate were assayed. Insulin-induced stimulation of L-dopa uptake was also abolished by inhibition of phosphatidylinositol 3-kinase (PI3K; 100 nM wortmannin, and 25 [micro]M LY-294002) and protein kinase C (PKC; 1 [micro]M RO-318220). Insulin-induced activation of PKC-[zeta] was confirmed in vitro by its translocation from the cytosol to the membrane fraction, and in vivo by immunohistochemistry studies. Insulin caused a wortmannin-sensitive increase in Akt/protein kinase B (Akt/PKB) phosphorylation and a dose-dependent translocation of Akt/PKB to the membrane fraction. Our findings suggest that insulin activates PKC-[zeta], and Akt/PKB downstream of PI3K, and that these pathways contribute to the insulin-induced increase of L-dopa uptake into PTCs. amino acid transport; second messengers; dopamine

This is the first description of functional immunoassay technology (FIT), which as a diagnostic tool has broad application across the whole spectrum of physiological measurements. In this paper, FIT is used to measure the renal clearance of an ultra low-dose administration of a clinically available contrast reagent for the purpose of obtaining an accurate glomerular filtration rate (GFR) measurement. Biomarker-based GFR estimates offer convenience, but are not accurate and are often misleading. FIT overcomes previous analytic barriers associated with obtaining an accurate GFR measurement. We present the performance characteristics of this diagnostic test and demonstrate the method by directly comparing GFR values obtained by FIT to those obtained by an FDA approved nuclear test in 20 adults. Two subjects were healthy volunteers and the remaining 18 subjects had diagnosed chronic kidney disease, with 12 being kidney transplant recipients. Measured GFR values were calculated by the classic UV/P method and by the blood clearance method. GFR obtained by FIT and the nuclear test correlated closely over a wide range of GFR values (10.9-102.1 ml x [min.sup.-1] x 1.73 [m.sup.-2]). The study demonstrates that FIT-GFR provides an accurate and reproducible measurement. This nonradioactive, immunoassay-based approach offers many advantages, chiefly that most laboratories already have the equipment and trained personnel necessary to run an ELISA, and therefore this important diagnostic measurement can more readily be obtained. The FIT-GFR test can be used throughout the pharmaceutical development pipeline: preclinical and clinical trials. renal function; chronic kidney disease; transplant

Activation of [mu]-opioid receptors makes animals hyperphagic and increases their preference for a high-fat diet. Previous studies have suggested that this receptor population plays a role in mediating the hyperphagia that is associated with food deprivation. In this paper, we tested the hypothesis that food deprivation will increase the expression of [mu]opioid receptors in the ventral medial hypothalamus and arcuate nucleus (VMH/ARC). Food deprivation resulted in a significant increase in the mRNA expression of [mu]-opioid receptors in the VMH/ARC and the lateral hypothalamus (LH) after 48 h of fasting but not after 24 or 12 h of fasting in either the light or dark. We did not observe a change in the mRNA expression of [kappa]- or [delta]-opioid receptors after food deprivation. When food-deprived animals were given a choice between a low-fat diet and a high-fat diet, they were hyperphagic and consumed significantly more of the high-fat diet. When the [mu]-opioid receptors were blocked with [beta]-funaltrexamine (selective [mu]-opioid receptor antagonist), prior to giving food-deprived animals access to both a low-fat and high-fat diet, it significantly decreased the percentage of high-fat diet consumed. These data demonstrate that hypothalamic [mu]-opioid receptors may contribute to the hyperphagia and increased preference for a high-fat diet that is associated with food deprivation. food preference; hyperphagia

A method for estimation of central arterial pressure based on linear one-dimensional wave propagation theory is presented in this paper. The equations are applied to a distributed model of the arterial tree, truncated by three-element windkessels. To reflect individual differences in the properties of the arterial trees, we pose a minimization problem from which individual parameters are identified. The idea is to take a measured waveform in a peripheral artery and use it as input to the model. The model subsequently predicts the corresponding waveform in another peripheral artery in which a measurement has also been made, and the arterial tree model is then calibrated in such a way that the computed waveform matches its measured counterpart. For the purpose of validation, invasively recorded abdominal aortic, brachial, and femoral pressures in nine healthy subjects are used. The results show that the proposed method estimates the abdominal aortic pressure wave with good accuracy. The root mean square error (RMSE) of the estimated waveforms was 1.61 [+ or -] 0.73 mmHg, whereas the errors in systolic and pulse pressure were 2.32 [+ or -] 1.74 and 3.73 [+ or -] 2.04 mmHg, respectively. These results are compared with another recently proposed method based on a signal processing technique, and it is shown that our method yields a significantly (P < 0.01) lower RMSE. With more extensive validation, the method may eventually be used in clinical practice to provide detailed, almost individual, specific information as a valuable basis for decision making. arterial tree; parameter identification; mechanical model; transfer function

In 1926, the famous American pediatric cardiologist, Dr. Helen B. Tanssig, observed that in situs inversus totalis (SIT) main gross anatomical structures and the deep muscle bundles of the ventricles were a mirror image of the normal structure, while the direction of the superficial muscle bundles remained unchanged (H. B. Taussig, Bull Johns Hopkins Hosp 39: 199-202, 1926). She and we wondered about the implication of this observation for left ventricular (LV) deformation in SIT. We used magnetic resonance tagging to obtain information on LV deformation, rotation, and torsion from a series of tagged images in five evenly distributed, parallel, short-axis sections of the heart of nine controls and eight persons with SIT without other structural (cardiac) defect. In the controls, during ejection, the apex rotated counterclockwise with respect to the base, when looking from the apex. Furthermore, the base-to-apex gradient in rotation (torsion) was negative and similar at all longitudinal levels of the LV. In SIT hearts, torsion was positive near the base, indicating mirrored myofiber orientations compared with the normal LV. Contrary to expectations, torsion in the apical regions of SIT LVs was as in normal ones, reflecting a normal internal myocardial architecture. The transition zone with zero torsion, found between the apex and base, suggests that the heart structure in SIT is essentially different from that in the normal heart. This provides a unique possibility to study regulatory mechanisms for myocardial fiber orientation and mechanical load, which has been dealt with in the companion paper by Kroon et al. cardiac development; adaptation; magnetic resonance imaging; mechanics; myocardium; ventricular function

Lipopmtein metabolism plays an important role in the development of several human diseases, including coronary artery disease and the metabolic syndrome. A good comprehension of the factors that regulate the metabolism of the various lipoproteins is therefore key to better understanding the variables associated with the development of these diseases. Among the players identified are regulators such as caveolins and caveolae. Caveolae are small plasma membrane invaginations that are observed in terminally differentiated cells. Their most important protein marker, caveolin-1, has been shown to play a key role in the regulation of several cellular signaling pathways and in the regulation of plasma lipoprotein metabolism. In the present paper, we have examined the role of caveolin-1 in lipoprotein metabolism using caveolin1-deficient (Cav-1-/-) mice. Our data show that, while Cav-l-/mice show increased plasma triglyceride levels, they also display reduced hepatic very low-density lipoprotein (VLDL) secretion. Additionally, we also found that a caveolin-1 deficiency is associated with an increase in high-density lipoprotein (HDL), and these HDL particles are enriched in cholesteryl ester in Cav-l-/- mice when compared with HDL obtained from wild-type mice. Finally, our data suggest that a caveolin-I deficiency prevents the transcytosis of LDL across endothelial cells, and therefore, that caveolin-1 may be implicated in the regulation of plasma LDL levels. Taken together, our studies suggest that caveolin-1 plays an important role in the regulation of lipoprotein metabolism by controlling their plasma levels as well as their lipid composition. Thus caveolin-1 may also play an important role in the development of atherosclerosis. cholesterol; high-density lipoprotein

Classical theories suggest that the surface area of the crystalline lens changes during accommodation while the lens volume remains constant. Our recent work challenged this view by showing that the lens volume decreases as the lens flattens during unaccommodation. In this paper we investigate 1) the magnitude of changes in the surface of the in vitro isolated cow lens during simulated accommodation, as well as that of human lens models, determined from lateral photographs and the application of the first theorem of Pappus; and 2) the velocity of the equatorial diameter recovery of prestretched cow and rabbit lenses by using a custom-built software-controlled stretching apparatus synchronized to a digital camera. Our results showed that the in vitro cow lens surface changed in an unexpected manner during accommodation depending on how much tension was applied to flatten the lens. In this case, the anterior surface initially collapsed with a reduction in surface followed by an increase in surface, when the stretching was applied. In the human lens model, the surface increased when the lens unaccommodated. The lens volume always decreases as the lens flattens. An explanation for the unexpected surface change is presented and discussed. Furthermore, we determined that the changes in lens volume, as reflected by the speed of the equatorial diameter recovery in in vitro cow and rabbit lenses during simulated accommodation, occurred within a physiologically relevant time frame (200 ms), implying a rapid movement of fluid to and from the lens during accommodation. lens surface area calculation; lens volume calculation; lenticular fluid movement; in vitro model for accommodation

The signal transducers and activators of transcription (STAT) family of transcription factors regulates a variety of biological functions including cellular proliferation, transformation, apoptosis, and differentiation. We have previously determined that PDGF activates the STAT pathway in human airway smooth muscle cells (HASMC) and that the Jak and Src kinases are required for both PDGF-induced STAT activation and HASMC proliferation. As increased airway smooth muscle (ASM) volume is associated with airflow obstruction and disease severity in patients with asthma, it is important to elucidate the cellular and molecular pathways that regulate ASM accumulation. In this paper, we investigated the requirement of STAT3 for PDGFinduced HASMC proliferation. We demonstrate that knockdown of STAT3 expression in HASMC resulted in a significant decrease in mitogen-induced cellular proliferation. Additionally, PDGF-induced activation of STAT3 required the small GTP-binding protein Rac1, and Rac1 was also required for PDGF-induced HASMC proliferation. Furthermore, PDGF treatment induced STAT3 and Rac1 to physically associate and translocate to the nucleus, identifying one mechanism by which STAT3 is regulated in response to PDGF in HASMC. Finally, we determined that STAT3 expression is required for PDGF-mediated regulation of cell cycle targets cyclin D3 and p27. These data define a novel mitogenic signaling pathway in airway smooth muscle cells leading from PDGF to Rac1 and STAT3 and subsequent cell cycle gene regulation. Thus, targeting STAT3 may prove to be a novel therapeutic approach for patients with severe asthma and significant airway wall remodeling, as manifested by ASM accumulation. asthma; airway remodeling; human airway smooth muscle cells

It is well known that the force developed by skeletal muscles increases with temperature. Despite the work done on this subject, the mechanism of force potentiation is still debated. Most of the published papers suggest that force enhancement is due to the increase of the individual cross-bridge force. However, reports on skinned fibers and single-molecule experiments suggest that cross-bridge force is temperature independent. The effects of temperature on cross-bridge properties in intact frog fibers were investigated in this study by applying fast stretches at various tension levels (P) on the tetanus rise at 5[degrees]C and 14[degrees]C to induce cross-bridge detachment. Cross-bridge number was measured from the force (critical force, [P.sub.c]) needed to detach the cross-bridge ensemble, and the average cross-bridge strain was calculated from the sarcomere elongation needed to reach [P.sub.c] (critical length, [L.sub.c]). Our results show that Pc increased linearly with the force developed at both temperatures, but the [P.sub.c]/P ratio was considerably smaller at 14[degrees]C. This means that the average force per cross bridge is greater at high temperature. This mechanism accounts for all the tetanic force enhancement. The critical length [L.sub.c] was independent of the tension developed at both temperatures but was significantly lower at high temperature suggesting that cross bridges at 14[degrees]C are more strained. The increased cross-bridge strain accounts for the greater average force developed. force enhancement; fast stretches

The ability of the distal lung epithelia to actively transport [Na.sup.+], with [Cl.sup.-] and water following, from the alveolar spaces inversely correlates with morbidity and mortality of infants, children, and adults with alveolar pulmonary edema. It is now recognized, in contrast to many other [Na.sup.+] transporting epithelia, that at least half of this active transport is not sensitive to amiloride, which inhibits the epithelial [Na.sup.+] channel. This paper reviews amiloride-insensitive [Na.sup.+] and fluid transport in the mammalian distal lung unit under basal conditions and speculates on potential explanations for this amiloride-insensitive transport. It also provides new information, using primary cultures of rat fetal distal lung epithelia and alveolar type II cells grown under submersion and air-liquid interlace culture conditions, regarding putative blockers of this transport. sodium

Unipolar electrograms (EGs) and hybrid (or unorthodox or unipolar) monophasic action potentials (HMAPs) are currently the only proposed extracellular electrical recording techniques for obtaining cardiac recovery maps with high spatial resolution in exposed and isolated hearts. Estimates of the repolarization times from the HMAP downstroke phase have been the subject of recent controversies. The goal of this paper is to computationally address the controversies concerning the HMAP information content, in particular the reliability of estimating the repolarization time from the HMAP downstroke phase. Three-dimensional numerical simulations were performed by using the anisotropic bidomain model with a region of short action potential durations. EGs, transmembrane action potentials (TAPs), and HMAPs elicited by an epicardial stimulation close or away from a permanently depolarized site were computed. The repolafization time was computed as the moment of EG fastest upstroke ([RT.sub.eg]) during the T wave, of HMAP fastest downstroke ([RT.sub.HMAP]), and of TAP fastest downstroke ([RT.sub.tap]). The latter was taken as the gold standard for repolarization time. We also compared the times ([RT90.sub.HMAP], [RT90.sub.tap]) when the HMAP and TAP first reach 90% of their resting value during the downstroke. For all explored sites, the HMAP downstroke closely followed the TAP downstroke, which is the expression of local repolarization activity. Results show that HMAP and TAP markers are highly correlated, and both markers [RT.sub.HMAP] and [RT.sub.eg] ([RT90.sub.HMAP]) are reliable estimates of the TAP reference marker [RT.sub.tap] ([RT90.sub.tap]). Therefore, the downstroke phase of the HMAP contains valuable information for assessing repolarization times. unipolar electrograms; monophasic action potential; bidomain model; heterogeneity; action potential duration

Epidemiological and animal studies suggest that the alteration of hormonal and metabolic environment during fetal and neonatal development can contribute to development of metabolic syndrome in adulthood. In this paper, we investigated the impact of maternal high-fat (HF) diet on hypothalamic leptin sensitivity and body weight gain of offspring. Adult Wistar female rats received a HF or a control normal-fat (C) diet for 6 wk before gestation until the end of the suckling period. After weaning, pups received either C or HF diet during 6 wk. Body weight gain and metabolic and endocrine parameters were measured in the eight groups of rats formed according to a postweaning diet, maternal diet, and gender. To evaluate hypothalamic leptin sensitivity in each group, STAT-3 phosphorylation was measured in response to leptin or saline intraperitoneal bolus. Pups exhibited similar body weights at birth, but at weaning, those born to HF dams weighed significantly less (-12%) than those born to C dams. When given the HF diet, males and females born to HF dams exhibited smaller body weight and feed efficiency than those born to C dams, suggesting increased energy expenditure programmed by the maternal HF diet. Thus, maternal HF feeding could be protective against adverse effects of the HF diet as observed in male offspring of control dams: overweight (+ 17%) with hyperleptinemia and hyper-insulinemia. Furthermore, offspring of HF dams fed either C or HF diet exhibited an alteration in hypothalamic leptin-dependent STAT-3 phosphorylation. We conclude that maternal high-fat diet programs a hypothalamic leptin resistance in offspring, which, however, fails to increase the body weight gain until adulthood. leptin resistance; hypothalamus; high-fat diet

We have previously shown that deficient arginine intake increased the rate of endogenous arginine synthesis from proline. In this paper, we report in vivo quantification of the effects of arginine intake on total endogenous arginine synthesis, on the rates of conversion between arginine, citrulline, ornithine, and proline, and on nitric oxide synthesis. Male piglets, with gastric catheters for diet and isotope infusion and femoral vein catheters for blood sampling, received a complete diet for 2 days and then either a generous (+Arg; 1.80 g*[kg.sup.-1]*[day.sup.-1]; n = 5) or deficient (-Arg; 0.20 g*[kg.sup.-1]*[day.sup.-1];n = 5) arginine diet for 5 days. On day 7, piglets received a primed, constant infusion of [[guanido-.sup.15][N.sub.2]]arginine, [[ureido-.sup.13]C;5,5-2H2]citrulline, [[U-.sup.13][C.sub.5]]omithine, and [[sup.15]N;[U.sup.13]Cs] proline in an integrated study of the metabolism of arginine and its precursors. Arginine synthesis ([micro]mol*[kg.sup.-1]*[h.sup.-1]) from both proline (+Arg: 42, -Arg: 74, pooled SE: 5) and citrulline (+Arg: 67, -Arg: 120; pooled SE: 15) were higher in piglets receiving the -Pug diet (P < 0.05); and for both diets proline accounted for ~60% of total endogenous arginine synthesis. The conversion of proline to citrulline (+Pug: 39, -Pug: 67, pooled SE: 6) was similar to the proline-to-arginine conversion, confirming that citrulline formation limits arginine synthesis from proline in piglets. Nitric oxide synthesis ([micro]mol*[kg.sup.-1]* [h.sup.-1]), measured by the rate conversion of [gua[nido-.sup.15][N.sub.a]]arginine to [[ureido-.sup.15]]citrulline, was greater in piglets receiving the +Pug diet (105) than in those receiving the -Pug diet (46, pooled SE: 10; P < 0.05). This multi-isotope method successfully allowed many aspects of arginine metabolism to be quantified simultaneously in vivo. arginine synthesis; nitric oxide; arginine precursors

Human organic anion transporter 1 (OAT1, SLC22A6), which is localized to the basolateral membranes of renal tubular epithelial cells, plays a critical role in the excretion of anionic compounds. OAT1 is regulated by various pathophysiological conditions, but little is known about the molecular mechanisms regulating the expression of OAT1. In the present study, we investigated the transcriptional regulation of OAT1 and found that hepatocyte nuclear factor (HNF)-4[alpha] markedly trans-activated the OAT1 promoter. A deletion analysis of the OAT1 promoter suggested that the regions spanning -1191 to -700 base pairs (bp) and -140 to -79 bp were essential for the transactivation by HNF-4[alpha]. These regions contained a direct repeat separated by two nucleotides (DR-2), which is one of the consensus sequences binding to HNF-4[alpha], and an inverted repeat separated by eight nucleotides (IR-8), which was recently identified as a novel element for HNF-4[alpha], respectively. An electrophoretic mobility shift assay showed that HNF-4[alpha] bound to DR-2 and IR-8 under the conditions of HNF-4[alpha] overexpression. Furthermore, under normal conditions, HNF-4[alpha] bound to IR-8, and a mutation in IR-8 markedly reduced the OAT1 promoter activity, indicating that HNF-4[alpha] regulates the basal transcription of OAT1 via IR-8. This paper reports the first characterization of the human OAT1 promoter and the first gene in the kidney whose promoter activity is regulated by HNF-4[alpha]. SLC22A6; proximal tubule; promoter; inverted repeat-8 doi:10.1152/ajprenal.00017.2007

Amiloride-sensitive [Na.sup.+] absorption is a well-described feature of numerous transporting epithelia in vertebrates. Yet, very little is known about this important physiological process regarding invertebrates. In the present paper, we compare vertebrate [Na.sup.+] absorption mediated by the amiloride-sensitive epithelial [Na.sup.+] channel (ENaC) and its invertebrate counterpart. We used the dorsal skin of the annelid Hirudo medicinalis as a model for the [Na.sup.+] absorption of invertebrate epithelia. In applying electrophysiological, molecular, and biochemical techniques we found striking functional and structural differences between vertebrate and invertebrate amiloride-sensitive [Na.sup.+] absorption. Using modified Ussing chambers, we analyzed the influence of different known blockers and effectors of vertebrate ENaC on leech epithelial [Na.sup.+] absorption. We demonstrate that the serine protease trypsin had no effect on the [Na.sup.+] transport across leech integument, while it strongly activates vertebrate ENaC. While protons, and the divalent cations [Ni.sup.2+] and [Zn.sup.2+] stimulate vertebrate ENaC, amiloride-sensitive [Na.sup.+] currents in leech integument were substantially reduced. For molecular studies, we constructed a cDNA library of Hirudo medicinalis and screened it with specific ENaC antibodies. We performed numerous PCR approaches using a vast number of different degenerated and specific ENaC primers to identify ENaC-like structures. Yet, both strategies did not reveal any ENaC-like sequence in leech integument. From these data we conclude that amiloride-sensitive [Na.sup.+] absorption in leech skin is not mediated by an ENaC-like [Na.sup.+] channel but by a still unknown invertebrate member of the ENaC/DEG family that we termed 1ENaTP (leech epithelial [Na.sup.+] transporting protein). epithelial [Na.sup.+] channel/degenerin superfamily; invertebrate epithelial transport doi:10.1152/ajpregu.00549.2006

This paper provides molecular evidence for a liver glyconeogenic pathway, that is, a concomitant activation of hepatic gluconeogenesis and glycogenesis, which could participate in the mechanisms that cope with amino acid excess in high-protein (HP) fed rats. This evidence is based on the concomitant upregulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression, downregulation of glucose 6-phosphatase catalytic subunit (G6PC1) gene expression, an absence of glucose release from isolated hepatocytes and restored hepatic glycogen stores in the fed state in HP fed rats. These effects are mainly due to the ability of high physiological concentrations of portal blood amino acids to counteract glucagon-induced liver G6PC 1 but not PEPCK gene expression. These results agree with the idea that the metabolic pathway involved in glycogen synthesis is dependent upon the pattern of nutrient availability. This nonoxidative glyconeogenic disposal pathway of gluconeogenic substrates copes with amino excess and participates in adjusting both amino acid and glucose homeostasis. In addition, the pattern of PEPCK and G6PC1 gene expression provides evidence that neither the kidney nor the small intestine participated in gluconeogenic glucose production under our experimental conditions. Moreover, the main glucose-6-phosphatase (G6Pase) isoform expressed in the small intestine is the ubiquitous isoform of G6Pase (G6PC3) rather than the G6PC 1 isoform expressed in gluconeogenic organs. high-protein diet; glyconeogenesis; glucose 6-phosphatase; phosphoenolpyruvate carboxykinase; liver

Retinoic acid (RA) is a signaling molecule in the morphogenesis of the mammary gland, modulating the expression of matrix metalloproteinases (MMPs). The aim of this paper was to study the role of RA during weaning, which consists of three events: apoptosis of the secretory cells, degradation of the extracellular matrix, and adipogenesis. CRABP II and CRBP-1 carrier proteins increased significantly during weaning compared with lactating glands but reverted to control values after the litter resuckled. The effects of RA are mediated by the nuclear receptors RAR[alpha], RAR[beta], RAR[gamma], and RXR[alpha], which underwent an increase in protein levels during weaning. In an attempt to elucidate the RAR[alpha]-dependent signaling pathway, ChIP assays were performed. The results showed the binding of RAR[alpha] to the MMP-9 promoter after 24- and 72-h weaning together with its coactivator p300; this fact could be responsible for the increase found in MMP-9 mRNA and protein levels in these conditions. Expression of related MMPs (MMP-2 and MMP-3) was also increased during weaning. Using gelatine zymography, we observed a time-dependent increase in active forms of MMP-9 and MMP-2. On the other hand, the inhibitor of MMPs, TIMP-1, was almost undetectable at 24- and 72-h weaning by Western blot. The role of retinoids in matrix remodeling is reinforced by the fact that administration of an acute dose of retinol palmitate to control lactating rats also induces MMP-9 expression. This emphasizes the importance of retinoids in vivo to regulate mammary gland involution. mammary gland involution; matrix metalloproteinase-9; stromelysin-1; retinoic acid; retinoid acid receptor-[alpha];

Insulin receptors are widely distributed in the kidney and affect multiple aspects of renal function. In the proximal tubule, insulin regulates volume and acid-base regulation through stimulation of the [Na.sup.+]/[H.sup.+] exchanger NHE3. This paper characterizes the signaling pathway by which insulin stimulates NHE3 in a cell culture model [opossum kidney (OK) cell]. Insulin has two distinct phases of action on NHE3. Chronic insulin (24 h) activates NHE3 through the classic phosphatidylinositol 3-kinase-serum- and glucocorticoid-dependent kinase 1 (PI3K-SGK1) pathway as insulin stimulates SGK1 phosphorylation and the insulin effect can be blocked by the PI3K inhibitor wortmannin or a dominant-negative SGK1. We showed that SGK1 transcript and protein are expressed in rat proximal tubule and OK cells. We previously showed that glucocorticoids augment the effect of insulin on NHE3 (Klisic J, Hu MC, Nief V, Reyes L, Fuster D, Moe OW, Ambuhl PM. Am J Physiol Renal Physiol 283: F532-F539, 2002). Part of this can be mediated via induction of SGK1 by glucocorticoids, and indeed the insulin effect on NHE3 can also be amplified by overexpression of SGK1. We next addressed the acute effect of insulin (1-2 h) on NHE3 by systematically examining the candidate signaling cascades and activation mechanisms of NHE3. We ruled out the PI3K-SGK1-Akt and TC10 pathways, increased surface NHE3, NHE3 phosphorylation, NHE3 association with calcineurin homologous protein 1 or megalin as mechanisms of acute activation of NHE3 by insulin. In summary, insulin stimulates NHE3 acutely via yet undefined pathways and mechanisms. The chronic effect of insulin is mediated by the classic PI3K-SGK1 route. [Na.sup.+]/[H.sup.+] exchanger isoform 3; serum- and glucocorticoid-dependent kinase 1

Potential functional consequences of the differences in protein distribution between the mitochondria of the rat liver, heart, brain, and kidney, as determined in the companion paper in this issue (Johnson DT, French S, Blair PV, You JS, Bemis KG, Wang M, Harris RA, and Balaban RS. The tissue heterogeneity of the mammalian mitochondrial proteome. Am J Physiol Cell Physiol 292: C689-C697, 2006), were analyzed using a canonical metabolic pathway approach as well as a functional domain homology analysis. These data were inserted into the Kyoto Encyclopedia of Genes and Genomes pathway framework to give global and metabolic pathway-specific information on the impact of the differential protein distribution on mitochondrial function. Custom pathway analysis was also performed using pathways limited to the mitochondrion. With the use of this approach, several well-known functional differences between these mitochondrial populations were confirmed. These included GABA metabolism in the brain, urea synthesis in the liver, and the domination of oxidative phosphorylation in the heart. By comparing relative protein amounts of mitochondria across tissues, a greater understanding of functional emphasis is possible as well as the nuclear 'programming' required to enhance a given function within the mitochondria. For proteins determined to be mitochondrial and lacking a defined role functional domain BLAST analyses were performed. Several proteins associated with DNA structural modification and a novel CoA transferase were identified. A protein was also identified capable of catalyzing the first three steps of de novo pyrimidine synthesis. This analysis demonstrates that the distribution of nuclear encoded proteins significantly modifies the overall functional emphasis of the mitochondria to meet tissue-specific needs. These studies demonstrate the existence of mitochondrial biochemical functions that at present are poorly defined. oxidative phosphorylation; liquid chromatography; mass spectrometry; electrophoresis; histone; liver; heart; kidney; brain

The extensive development of detailed mathematical models of cardiac myocyte electrophysiology in recent years has led to a proliferation of models, including many that model the same animal species and specific region of the heart and thus would be expected to have similar properties. In this paper we review and compare two recently developed mathematical models of the electrophysiology of canine ventricular myocytes. To clarify their similarities and differences, we also present studies using them in a range of preparations from single cells to two-dimensional tissue. The models are compared with each other and with new and previously published experimental results in terms of a number of their properties, including action potential morphologies; transmembrane currents during normal heart rates and during alternans; alternans onsets, magnitudes, and cessations; and reentry dynamics of spiral waves. Action potential applets and spiral wave movies for the two canine ventricular models are available online as supplemental material. We find a number of differences between the models, including their rate dependence, alternans dynamics, and reentry stability, and a number of differences compared with experiments. Differences between models of the same species and region of the heart are not unique to these canine models. Similar differences can be found in the behavior of two models of human ventricular myocytes and of human atrial myocytes. We provide several possible explanations for the differences observed in models of the same species and region of the heart and discuss the implications for the applicability of models in addressing questions of mechanism in cardiac electrophysiology. ionic models; ventricular arrhythmias; electrical alternans

Myofibrillar protein loss occurring in catabolic situations is considered to be mediated by the release of proinflammatory cytokines and associated with a decrease in circulating and muscle levels of insulin-like growth factor I (IGF-I). In this paper, we investigated whether the [C.sub.2][C.sub.12] myotube atrophy caused in vitro by TNF-[alpha]/IFN-[gamma] cytokines might be reversed by exogenous IGF-I. Our results showed that, despite the presence of TNF-[alpha]/IFN-[gamma], IGF-I retained its full ability to induce the phosphorylation of Akt, Foxo3a, and GSK-3[beta] (respectively, 16-fold, 9-fold, and 2-fold) together with a decrease in atrogin-1 mRNA (-39%, P < 0.001). Although this ubiquitin ligase has been reported to accelerate the degradation of MyoD, a myogenic transcription factor driving the transcription of myosin heavy chain (MHC), IGF-I failed to blunt the reduction of MyoD and MHC caused by TNF-[alpha]/IFN-[gamma]. Moreover, IGF-I only very slightly attenuated the myotube atrophy induced by TNF-[alpha]/IFN-[gamma] (TNF-[alpha]/IFN-[gamma] 15.48 [micro]m alone vs. TNF-[alpha]/IFN-[gamma]/IGF-I 16.97 [micro]m, P < 0.001). In conclusion, our data show that IGF-I does not reverse the myotube atrophy induced by TNF-[alpha]/IFN-[gamma] despite the phosphorylation of Foxo and GSK-3[beta] and the downregulation of atrogin-1 mRNA. Our study suggests therefore that factors other than IGF-I decrease are responsible for the muscle atrophy caused by proinflammatory cytokines. growth factors; ubiquitin-proteasome system; tumor necrosis factor-[alpha]; transduction pathway; muscle atrophy; glycogen synthase kinase-3[beta]

Statistical analysis of time series is still inadequate within circulation research. With the advent of increasing computational power and real-time recordings from hemodynamic studies, one is increasingly dealing with vast amounts of data in time series. This paper aims to illustrate how statistical analysis using the significant nonstationarities (SiNoS) method may complement traditional repeated-measures ANOVA and linear mixed models. We applied these methods on a dataset of local hepatic and systemic circulatory changes induced by aortoportal shunting and graded liver resection. We found SiNoS analysis more comprehensive when compared with traditional statistical analysis in the following four ways: 1) the method allows better signal-to-noise detection; 2) including all data points from real time recordings in a statistical analysis permits better detection of significant features in the data; 3) analysis with multiple scales of resolution facilitates a more differentiated observation of the material; and 4) the method affords excellent visual presentation by combining group differences, time trends, and multiscale statistical analysis allowing the observer to quickly view and evaluate the material. It is our opinion that SiNoS analysis of time series is a very powerful statistical tool that may be used to complement conventional statistical methods. statistical analysis; repeated measures

Ventricular fibrillation (VF) is one of the main causes of death in the Western world. According to one hypothesis, the chaotic excitation dynamics during VF are the result of dynamical instabilities in action potential duration (APD) the occurrence of which requires that the slope of the APD restitution curve exceeds 1. Other factors such as electrotonic coupling and cardiac memory also determine whether these instabilities can develop. In this paper we study the conditions for altemans and spiral breakup in human cardiac tissue. Therefore, we develop a new version of our human ventricular cell model, which is based on recent experimental measurements of human APD restitution and includes a more extensive description of intracellular calcium dynamics. We apply this model to study the conditions for electrical instability in single cells, for reentrant waves in a ring of cells, and for reentry in two-dimensional sheets of ventricular tissue. We show that an important determinant for the onset of instability is the recovery dynamics of the fast sodium current. Slower sodium current recovery leads to longer periods of spiral wave rotation and more gradual conduction velocity restitution, both of which suppress restitution-mediated instability. As a result, maximum restitution slopes considerably exceeding 1 (up to 1.5) may be necessary for electrical instability to occur. Although slopes necessary for the onset of instabilities found in our study exceed 1, they are within the range of experimentally measured slopes. Therefore, we conclude that steep APD restitution-mediated instability is a potential mechanism for VF in the human heart. reentrant arrhythmias; human ventricular myocytes; restitution properties; spiral waves; computer simulation doi:10.1152/ajpheart.00109.2006

The periaqueductal gray (PAG) is an important integrative region in the regulation of autonomic outflow and cardiovascular function and may serve as a regulatory center as part of a long-loop pathway during somatic afferent stimulation with acupuncture. Because the ventrolateral PAG (vlPAG) provides input to the rostral ventrolateral medulla (rVLM), an important area for electroacupuncture (EA) regulation of sympathetic outflow, we hypothesized that the vlPAG plays a role in the EA-related modulation of rVLM premotor sympathetic neurons activated during visceral afferent stimulation and autonomic excitatory reflexes. Cats were anesthetized and ventilated, and heart rate and mean blood pressure were monitored. Stimulation of the splanchnic nerve by a pledget of filter paper soaked in bradykinin (BK, 10 [micro]g/ml) every 10 min on the gallbladder induced consistent cardiovascular reflex responses. Bilateral stimulation with EA at acupoints over the pericardial meridian (P5-6) situated over the median nerve reduced the increases in blood pressure from 34 [+ or -] 3 to 18 [+ or -] 5 mmHg for a period of time that lasted for 60 min or more. Unilateral inactivation of neuronal activity in the vlPAG with 50-75 nl of kainic acid (KA, 1 mM) restored the blood pressure responses from 18 [+ or -] 3 to 36 [+ or -] 5 mmHg during BK-induced gallbladder stimulation, an effect that lasted for 30 min. In the absence of EA, unilateral microinjection of the excitatory amino acid DL-homocysteic acid (DLH, 4 nM) in the vlPAG mimicked the effect of EA and reduced the reflex blood pressure responses from 35 [+ or -] 6 to 14 [+ or -] 5 mmHg. Responses of 21 cardiovascular sympathoexcitatory rVLM neurons, including 12 that were identified as premotor neurons, paralleled the cardiovascular responses. Thus splanchnic nerve-evoked neuronal discharge of 32 [+ or -] 4 spikes/30 stimuli in six neurons was reduced to 10 [+ or -] 2 spikes/30 stimuli by EA, which was restored rapidly to 28 [+ or -] 4 spikes/30 stimuli by unilateral injection of 50 nl KA into the vlPAG. Conversely, 50 nl of DLH in the vlPAG reduced the number of action potentials of 5 rVLM neurons from 30 [+ or -] 4 to 18 [+ or -] 4 spikes/30 stimuli. We conclude that the inhibitory influence of EA involves vlPAG stimulation, which, in turn, inhibits rVLM neurons in the EA-related attenuation of the cardiovascular excitatory response during visceral afferent stimulation. somatic afferents; premotor sympathetic; visceral afferents; Neiguan-Jianshi acupoints

Toll-like receptors (TLRs) are now recognized as the major receptors for microbial pathogens on cells of the innate immune system. Recently, TLRs were also identified in many organs including the kidney. However, the cellular distribution and role of these renal TLRs remain largely unknown. In this paper, we investigated the expression of TLR4 in a cecal ligation and puncture (CLP) model of sepsis in Sprague-Dawley rats utilizing fluorescence microscopy. In sham animals, TLR4 was expressed predominantly in Tamm-Horsfall protein (THP)-positive tubules. In CLP animals, TLR4 expression increased markedly in all tubules (proximal and distal), glomeruli, and the renal vasculature. The staining showed a strong apical distribution in all tubules. A moderately less intense cellular signal colocalized partially with the Golgi apparatus. In addition, kidneys from septic rats showed increased expression of CD14 and THP. They each colocalized strongly with TLR4, albeit in different tubular segments. We also imaged the kidneys of live septic animals with two-photon microscopy after fluorescent lipopolysaccharide (LPS) injection. Within 10 min, LPS was seen at the brush border of some proximal tubules. Within 60 min, LPS was fully cytoplasmic in proximal tubules. Conversely, distal tubules showed no LPS uptake. We conclude that TLR4, CD14, and THP have specific renal cellular and tubular expression patterns that are markedly affected by sepsis. Systemic endotoxin can freely access the tubular and cellular sites where these proteins are present. Therefore, locally expressed TLRs and other interacting proteins could potentially modulate the renal response to systemic sepsis. TLR4; CD14; Tamm-Horsfall protein; cecal ligation and puncture: lipopolysaccharide

Bright light can influence human psychophysiology instantaneously by inducing endocrine (suppression of melatonin, increasing cortisol levels), other physiological changes (enhancement of core body temperature), and psychological changes (reduction of sleepiness, increase of alertness). Its broad range of action is reflected in the wide field of applications, ranging from optimizing a work environment to treating depressed patients. For optimally applying bright light and understanding its mechanism, it is crucial to know whether its effects depend on the time of day. In this paper, we report the effects of bright light given at two different times of day on psychological and physiological parameters. Twenty-four subjects participated in two experiments (n = 12 each). All subjects were nonsmoking, healthy young males (18-30 yr). In both experiments, subjects were exposed to either bright light (5,000 lux) or dim light < 10 lux (control condition) either between 12:00 P.M. and 4:00 P.M. (experiment A) or between midnight and 4:00 A.M. (experiment B). Hourly measurements included salivary cortisol concentrations, electrocardiogram, sleepiness (Karolinska Sleepiness Scale), fatigue, and energy ratings (Visual Analog Scale). Core body temperature was measured continuously throughout the experiments. Bright light had a time-dependent effect on heart rate and core body temperature; i.e., bright light exposure at night, but not in daytime, increased heart rate and enhanced core body temperature. It had no significant effect at all on cortisol. The effect of bright light on the psychological variables was time independent, since nighttime and daytime bright light reduced sleepiness and fatigue significantly and similarly. sleepiness; core body temperature; cortisol; heart rate

Thyrotropin-releasing hormone (TRH) and its receptor subtype TRH receptor-1 (TRHR1) are found in pancreatic [beta]-cells, and it has been shown that TRH might have potential for autocrine/paracrine regulation through the TRHR1 receptor. In this paper, TRHR1 is studied to find whether it can initiate multiple signal transduction pathways to activate the epidermal growth factor (EGF) receptor in pancreatic [beta]-cells. By initiating TRHR1 G protein-coupled receptor (GPCR) and dissociated [alpha][beta][lambda]-complex, TRH (200 nM) activates tyrosine residues at [Tyr.sup.845] (a known target for Src) and [Tyr.sup.01068] in the EGF receptor complex of an immortalized mouse [beta]-cell line, [beta]TC-6. Through manipulating the activation of Src, PKC, and heparin-binding EGF-like growth factor (HB-EGF), with corresponding individual inhibitors and activators, multiple signal transduction pathways linking TRH to EGF receptors in [beta]TC-6 cell line have been revealed. The pathways include the activation of Stc kinase and the release of HB-EGF as a consequence of matrix metalloproteinase (MMP)-3 activation. Alternatively, TRH inhibited PKC activity by reducing the EGF receptor serine/threonine phosphorylation, thereby enhancing tyrosine phosphorylation. TRH receptor activation of Src may have a central role in mediating the effects of TRH on the EGF receptor. The activation of the EGF receptor by TRH in multiple circumstances may have important implications for pancreatic [beta]-cell biology. heparin-binding epidermal growth factor-like growth factor; Src; protein kinase C; G protein; signal transduction

Regular physical exercise has beneficial effects in many human disease states, including cardiovascular diseases, cancer, and depression. Exercise training of genetically modified mouse models may provide insight into the molecular mechanisms that underlie the beneficial effects of exercise. Presently, there is relatively little understanding of the normal physiology of mouse exercise. In this paper, we describe a novel computerized voluntary wheel-running system capable of recording and analyzing individual wheel rotations. Using this system, we demonstrate that C57BL/6 mice run considerable distances during the night in short bouts and at a preferred speed: the cruising speed. We find that the vast majority of running occurs around this cruising speed, which is close to the maximum speed at which the animal can run but is significantly higher than the average speeds recorded by simple digital odometers. We describe how these parameters vary with exercise training and demonstrate marked sex differences in the patterns of voluntary exercise. The results of this study have important implications for the design and interpretation of both voluntary and forced exercise experiments in mouse models. The novel parameters described provide more physiological quantitative measures of voluntary exercise activity and training and will extend the physiological utility of exercise training as a phenotyping tool in genetic mouse models. voluntary exercise; computerized monitoring; circadian rhythm: physiological hypertrophy