bluuebunny/crossref_metadata_2025 huggingface dataset:
Signals mediating cleavage of intercellular adhesion molecule-1
Authors: Tsakadze Nina L. , Sen Utpal , Zhao Zhendong , Sithu Srinivas D. , English William R. , D'Souza Stanley E.
Published: February 2004

ICAM-1, a membrane-bound receptor, is released as soluble ICAM-1 in inflammatory diseases. To delineate mechanisms regulating ICAM-1 cleavage, studies were performed in endothelial cells (EC), human embryonic kidney (HEK)-293 cells transfected with wild-type (WT) ICAM-1, and ICAM-1 containing single tyrosine-to-alanine substitutions (Y474A, Y476A, and Y485A) in the cytoplasmic region. Tyrosine residues at 474 and 485 become phosphorylated upon ICAM-1 ligation and associate with signaling modules. Cleavage was assessed by using an antibody against the cytoplasmic tail of ICAM-1, which recognizes intact ICAM-1 and the 7-kDa membrane-bound fragment remaining after cleavage. Cleavage in HEK-293 WT cells was accelerated by phorbol ester PMA, whereas in EC it was induced by tumor necrosis factor-α. In both cell types, a 7-kDa ICAM-1 remnant was detected. Tyrosine phosphatase inhibitors dephostatin and sodium orthovanadate augmented cleavage. PD-98059 (MEK kinase inhibitor), geldanamycin and PP2 (Src kinase inhibitors), and wortmannin (phosphatidylinositol 3-kinase inhibitor) dose-dependently inhibited cleavage in both cell types. SB-203580 (p38 inhibitor) was more effective in EC, and D609 (PLC inhibitor) mostly affected cleavage in HEK-293 cells. Cleavage was drastically decreased in Y474A and Y485A, whereas it was marginally reduced in Y476A. Surprisingly, phosphorylation was not detectable on the 7-kDa fragment of ICAM-1. These results implicate distinct pathways in the cleavage process and suggest a preferred signal transmission route for ICAM-1 shedding in the two cell systems tested. Tyrosine residues Y474 and Y485 within the cytoplasmic sequence of ICAM-1 regulate the cleavage process.
Altered triglyceride-rich lipoprotein production in Zucker diabetic fatty rats
Authors: Chirieac Doru V. , Collins Heidi L. , Cianci Joanne , Sparks Janet D. , Sparks Charles E.
Published: February 2004

Triglyceride-rich lipoprotein (TRL) production was studied in Zucker diabetic fatty (ZDF) rats, a model of insulin-resistant type 2 diabetes progression. TRL production was measured in vivo by blocking catabolism with Triton WR-1339. Ten-week ZDF rats are hyperinsulinemic with increased TRL production [both triglyceride and apolipoprotein B (apoB)]. Twenty-week ZDF rats are insulinopenic, and TRL production is similar to lean controls. Insulin infusion suppresses glucose and free fatty acids in 10- and 20-wk ZDF rats. Increased TRL production is not reduced by insulin in 10-wk rats; however, at 20 wk, TRL production is suppressed by insulin. In vitro studies with hepatocytes derived from 10-wk ZDF rats showed minimal insulin dose effects on apoB secretion compared with the response and sensitivity of hepatocytes derived from 20-wk ZDF and control lean rats. Hepatic sterol regulatory-binding protein (SREBP)-1c mRNA levels are increased at 10 wk but return to control levels at 20 wk. ApoB mRNA levels are similar to lean controls at 10 and 20 wk. The following two mechanisms for hypertriglyceridemia associated with hyperinsulinemia are suggested: increased TRL synthesis and loss of TRL suppression. Increased triglyceride production in hyperinsulinemic rats likely relates to increased expression of SREBP-1c, whereas increased apoB production involves posttranscriptional processes.
Tissue-specific regulation of cytochromecoxidase subunit expression by thyroid hormone
Authors: Sheehan Treacey E. , Kumar Ponni A. , Hood David A.
Published: February 2004

The influence of thyroid hormone (T3) on respiration is partly mediated via its effect on the cytochrome c oxidase (COX) enzyme, a multi-subunit complex within the mitochondrial respiratory chain. We compared the expression of COX subunits I, III, Vb, and VIc and thyroid receptors (TR)α1 and TRβ1 with functional changes in COX activity in tissues that possess high oxidative capacities. In response to 5 days of T3treatment, TRβ1 increased 1.6-fold in liver, whereas TRα1 remained unchanged. T3also induced concomitant increases in the protein and mRNA expression of nuclear-encoded subunit COX Vb in liver, matched by a 1.3-fold increase in binding to a putative thyroid response element (TRE) within the COX Vb promoter in liver, suggesting transcriptional regulation. In contrast, T3had no effect on COX Vb expression in heart. T3produced a significant increase in COX III mRNA in liver but decreased COX III mRNA in heart. These changes were matched by parallel alterations in mitochondrial transcription factor A expression in both tissues. In contrast, COX I protein increased in both liver and heart 1.7- and 1.5-fold ( P < 0.05), respectively. These changes in COX I closely paralleled the T3-induced increases in COX activity observed in both of these tissues. In liver, T3induced a coordinated increase in the expression of the nuclear (COX Vb) and mitochondrial (COX I) genomes at the protein level. However, in heart, the main effect of T3was restricted to the expression of mitochondrial DNA subunits. Thus our data suggest that T3regulates the expression of COX subunits by both transcriptional and posttranscriptional mechanisms. The nature of this regulation differs between tissues possessing a high mitochondrial content, like liver and heart.
Hypoglycemia and the sympathoadrenal system: neurogenic symptoms are largely the result of sympathetic neural, rather than adrenomedullary, activation
Authors: DeRosa Michael A. , Cryer Philip E.
Published: February 2004

The relative contributions of the sympathetic nervous system and the adrenal medullae, the two components of the sympathoadrenal system, to the manifestations of hypoglycemia are largely unknown. We tested the hypothesis that the neurogenic symptoms of hypoglycemia are largely the result of sympathetic neural activation. To do so, we quantitated neurogenic symptoms, as well as norepinephrine (NE) kinetics and selected hemodynamic changes, during hyperinsulinemic euglycemic and stepped hypoglycemic clamps in 15 healthy control subjects (Controls) and four bilaterally adrenalectomized patients (ADX). Plasma epinephrine responses to hypoglycemia were virtually absent in ADX, as expected. Neurogenic symptom scores increased to higher values during the hypoglycemic compared with the euglycemic clamps in both Controls ( P < 0.0001) (e.g., final scores of 7.8 ± 1.2 vs. 3.0 ± 0.7) and ADX ( P < 0.0001) (e.g., final scores of 10.8 ± 4.1 vs. 2.5 ± 1.0). Plasma NE concentrations ( P < 0.0001) and systemic NE spillover ( P = 0.0007) increased during the hypoglycemic compared with the euglycemic clamps in Controls but not in ADX. Similarly, heart rate increased ( P = 0.0104), diastolic blood pressure decreased ( P = 0.0003), and forearm blood flow increased ( P < 0.0001) during the hypoglycemic compared with the euglycemic clamps in Controls but not in ADX. These data indicate that the neurogenic symptoms of hypoglycemia are largely the result of sympathetic neural, rather than adrenomedullary, activation. They also suggest that the plasma NE and hemodynamic responses to hypoglycemia are largely the result of adrenomedullary, rather that sympathetic neural, activation.
Subcutaneous abdominal preadipocyte differentiation in vitro inversely correlates with central obesity
Authors: Permana Paska A. , Nair Saraswathy , Lee Yong-Ho , Luczy-Bachman Georgia , Vozarova de Courten Barbora , Tataranni P. Antonio
Published: February 2004

Expansion of adipose tissue mass results from increased number and size of adipocyte cells. We hypothesized that subcutaneous abdominal preadipocytes in obese individuals might have an intrinsically higher propensity to differentiate into adipocytes. Thus we investigated the relationship between obesity and the level of in vitro preadipocyte differentiation in Pima Indians. Subcutaneous abdominal stromal vascular fractions containing preadipocytes were cultured from 58 nondiabetic subjects [31 M/27 F, 30 ± 6 yr, body fat 34 ± 8% by dual-energy X-ray absorptiometry (means ± SD)]. The average percentage of preadipocyte differentiation (PDIFF; cell count by microscopy) was 11 ± 11% (range 0.2–51%). PDIFF correlated negatively with percent body fat ( r = −0.35, P = 0.006) and waist circumference ( r = −0.45, P = 0.0004). Multiple regression analysis indicated that waist circumference ( P = 0.01), sex ( P = 0.01), and percent body fat ( P = 0.05) were significant determinants of PDIFF. Molecular characterization of predifferentiated cultured cells was performed by real-time PCR measurements of glucocorticoid receptor-α (GRα), insulin-like growth factor I receptor (IGF-IR), peroxisome proliferator-activated receptor-γ (PPARγ), enhancer-binding protein GATA-3, CCAAT/enhancer-binding protein-α undifferentiated protein (CUP/AP-2α), and endothelial cell-specific marker 2 (ECSM2). The mRNA concentrations of GRα correlated with PDIFF ( r = 0.29, P = 0.03), but the others did not (IGF-IR, r = 0.003, P = 1.0; PPARγ, r = −0.1, P = 0.5; GATA-3, r = 0.02, P = 0.9; CUP/AP-2α, r = −0.2, P = 0.1; ECSM2, r = 0.04, P = 0.7). Contrary to our hypothesis, the results may indicate a blunted in vitro differentiation potential of preadipocytes in centrally obese individuals. The lower differentiation potential of preadipocytes in the obese subjects might be due, at least partly, to decreased glucocorticoid receptor expression.
Regulation by glucose and calcium of the carboxylmethylation of the catalytic subunit of protein phosphatase 2A in insulin-secreting INS-1 cells
Authors: Palanivel Rengasamy , Veluthakal Rajakrishnan , Kowluru Anjaneyulu
Published: February 2004

Previously, we reported that the catalytic subunit of protein phosphatase 2A (PP2Ac) undergoes carboxylmethylation (CML) at its COOH-terminal leucine, and that inhibitors of such a posttranslational modification markedly attenuate nutrient-induced insulin secretion from isolated β-cells. More recent studies have suggested direct inhibitory effects of glucose metabolites on PP2A activity in isolated β-cells, implying that inhibition of PP2A leads to stimulation of insulin secretion. Because the CML of PP2Ac has been shown to facilitate the holoenzyme assembly and subsequent functional activation of PP2A, we investigated putative regulation by glucose of the CML of PP2Ac in insulin-secreting (INS)-1 cells. Our data indicated a marked inhibition by specific intermediates of glucose metabolism (e.g., citrate and phospho enolpyruvate) of the CML of PP2Ac in INS-1 cell lysates. Such inhibitory effects were also demonstrable in intact cells by glucose. Mannoheptulose, an inhibitor of glucose metabolism, completely prevented inhibitory effects of glucose on the CML of PP2Ac. Moreover, glucose-mediated inhibition of the CML of PP2Ac was resistant to diazoxide, suggesting that glucose metabolism and the generation of glucose metabolites might control inhibition of the CML of PP2Ac. A membrane-depolarizing concentration of KCl also induced inhibition of the CML of PP2Ac in intact INS cells. On the basis of these data, we propose that glucose metabolism and increase in intracellular calcium facilitate inhibition of the CML of PP2Ac, resulting in functional inactivation of PP2A. This, in turn, might retain the key signaling proteins of the insulin exocytotic cascade in their phosphorylated state, leading to stimulated insulin secretion.
Cyclopiazonic acid decreases spontaneous transient depolarizations in guinea pig mesenteric lymphatic vessels in endothelium-dependent and -independent manners
Authors: Ferrusi Ilia , Zhao Jun , van Helden Dirk , von der Weid Pierre-Yves
Published: February 2004

Guinea pig mesenteric lymphatic vessels exhibit vasomotion through a pacemaker mechanism that involves intracellular Ca2+release and resultant spontaneous transient depolarizations (STDs) of the smooth muscle membrane potential. This study presents a detailed characterization of the effects of cyclopiazonic acid (CPA) on this pacemaker activity. Microelectrode recordings from smooth muscle in vessel segments revealed that application of CPA (1–10 μM) caused a hyperpolarization accompanied by a decrease in the frequency and amplitude of STDs. The CPA-induced hyperpolarization was abolished after destruction of the endothelium and in the presence of NG-nitro-l-arginine (100 μM) or 1 H-[1,2,4]oxadiazolol-[4,3- a]quinoxaline-1-one (10 μM), which suggests a contribution of endothelium-derived nitric oxide (EDNO) in this response. In the absence of EDNO-induced effects, CPA decreased the frequency and amplitude of STDs recorded before and in the presence of the thromboxane A2mimetic U-46619, norepinephrine, or thimerosal. CPA abolished U-46619-induced vasomotion as determined by measurement of constriction-associated intracellular Ca2+concentration using the ratiometric Ca2+indicator fura-2. The endothelial actions of CPA were compared with those of ACh, which is known to cause EDNO release in this preparation. Although CPA and ACh both increased endothelial intracellular Ca2+concentration and depolarized the membrane potential, the kinetics of action for both parameters were markedly slower for CPA than ACh. These results suggest that CPA first hyperpolarizes the lymphatic smooth muscle and decreases STD frequency and amplitude through endothelial release of EDNO, and second, consistent with the action of CPA to inhibit sarcoplasmic reticulum Ca2+-ATPase and deplete Ca2+stores, it further reduces STD activity. Inhibition of the lymphatic smooth muscle pacemaker mechanism is thought to abolish agonist-induced vasomotion.
Caspase-dependent cytochrome c release and cell death in chick cardiomyocytes after simulated ischemia-reperfusion
Authors: Qin Yimin , Vanden Hoek Terry L. , Wojcik Kim , Anderson Travis , Li Chang-Qing , Shao Zuo-Hui , Becker Lance B. , Hamann Kimm J.
Published: February 2004

We recently demonstrated that reperfusion rapidly induces the mitochondrial pathway of apoptosis in chick cardiomyocytes after 1 h of simulated ischemia. Here we tested whether ischemia-reperfusion (I/R)-induced apoptosis could be initiated by caspase-dependent cytochrome c release in this model of cardiomyocyte injury. Fluorometric assays of caspase activity showed little, if any, activation of caspases above baseline levels induced by 1 h of ischemia alone. However, these assays revealed rapid activation of caspase-2, yielding a 2.95 ± 0.52-fold increase (over ischemia only) within the 1st h of reperfusion, whereas activities of caspases-3, -8, and -9 increased only slightly from their baseline levels. The rapid and prominent activation of caspase-2 suggested that it could be an important initiator caspase in this model, and using specific caspase inhibitors given only at the point of reperfusion, we tested this hypothesis. The caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(Ome)-Val-Ala-Asp(Ome)-CH2F was the only caspase inhibitor that significantly inhibited cytochrome c release from mitochondria. This inhibitor also completely blocked activation of caspases-3, -8, and -9. The caspase-3/7 inhibitor transiently and only partially blocked caspase-2 activity and was less effective in blocking the activities of caspases-8 and -9. The caspase-8 inhibitor failed to significantly block caspase-2 or -3, and the caspase-9 inhibitor blocked only caspase-9. Furthermore, the caspase-2 inhibitor protected against I/R-induced cell death, but the caspase-8 inhibitor failed to do so. These data suggest that active caspase-2 initiates cytochrome c release after reperfusion and that it is critical for the I/R-induced apoptosis in this model.
Gender differences in myogenic tone in superoxide dismutase knockout mouse: animal model of oxidative stress
Authors: Veerareddy Sukrutha , Cooke Christy-Lynn M. , Baker Philip N. , Davidge Sandra T.
Published: February 2004

Oxidative stress mediated by prooxidants has been implicated in the pathogenesis of vascular disorders. However, the effect of prooxidants on myogenic regulation of vascular function and the differential influence of gender is not known. SOD, an intracellular enzyme, restricts excess prooxidant levels and may limit vascular dysfunction. We therefore tested the effects of Cu,Zn SOD deficiency on vascular tone in both male and female SOD knockout (SOD−/−) mice. We hypothesized that myogenic tone would be enhanced in SOD−/− mice by excess prooxidants compared with wild-type control mice. Indeed, resistance-sized mesenteric arteries from SOD−/− mice exhibited enhanced myogenic tone compared with control mice. Myogenic tone was lower in female than male control mice. Interestingly, this gender effect was absent in SOD−/− mice, such that myogenic tone of mesenteric arteries from females was equated to that of arteries from males. Furthermore, the pathways that modulate myogenic tone were diverse. In both male and female control mice, inhibition of prostaglandin H synthase (PGHS) and nitric oxide synthase (NOS) pathways enhanced myogenic tone. In female SOD−/− mice, inhibition of PGHS and NOS pathways enhanced myogenic tone to a greater extent compared with control mice. Conversely, in male SOD−/− mice, NOS and PGHS inhibition did not alter tone and only inhibition of gap junctions enhanced myogenic tone. In conclusion, this study revealed enhanced myogenic tone in SOD−/− mice compared with control mice. Furthermore, Cu,Zn SOD deficiency particularly enhanced myogenic tone in female mice such that their vascular tone attained the level of male SOD−/− mice, possibly mediated by prooxidants.
Carboxypeptidase-mediated enhancement of nitric oxide production in rat lungs and microvascular endothelial cells
Authors: Hadkar Vaishali , Sangsree Sakonwun , Vogel Stephen M. , Brovkovych Viktor , Skidgel Randal A.
Published: February 2004

Membrane-bound regulatory carboxypeptidases cleave only COOH-terminal basic residues from peptides and proteins. To investigate whether carboxypeptidase-generated arginine can increase nitric oxide (NO) synthesis we perfused rat lungs from animals challenged with LPS or used rat lung microvascular endothelial cells (RLMVEC) stimulated with LPS and IFN-γ, conditions that induced inducible NO synthase (iNOS) expression. Addition of carboxypeptidase substrate furylacryloyl-Ala-Arg (Fa-A-R) or Arg to the lung perfusate increased NO production two- to threefold. The carboxypeptidase inhibitor 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA) blocked the effect of Fa-A-R but not free Arg. Lysine, an Arg transport inhibitor, blocked the increase in NO stimulated by Fa-A-R. HPLC analysis showed that Fa-A-R hydrolysis was blocked by MGTA but not lysine. In cytokine-treated RLMVEC, Fa-A-R also stimulated NO production inhibited by MGTA or lysine. Membrane fractions from rat lungs or RLMVEC contained carboxypeptidase M-like activity at neutral pH that increased twofold in RLMVEC treated with LPS + IFN-γ. The kinetics of NO production in RLMVEC was measured with a porphyrinic microsensor. Addition of 1 mM Arg or Fa-A-R to cells preincubated in Arg-free medium resulted in a slowly rising, prolonged (>20 min) NO output. NO production stimulated by Fa-A-R was blocked by MGTA or iNOS inhibitor 1400W. HPLC analysis of Fa-A-R hydrolysis revealed only 3.7 μM Arg was released over 20 min. Thus NO production in RLMVEC is stimulated more efficiently by Arg released from carboxypeptidase substrates than free Arg. These studies reveal a novel mechanism by which the Arg supply for NO production in inflammatory conditions may be maintained.
Altered bleomycin-induced lung fibrosis in osteopontin-deficient mice
Authors: Berman Jeffrey S. , Serlin David , Li Xinfang , Whitley Geoffrey , Hayes John , Rishikof David C. , Ricupero Dennis A. , Liaw Lucy , Goetschkes Margaret , O'Regan Anthony W.
Published: February 2004

Osteopontin is a multifunctional matricellular protein abundantly expressed during inflammation and repair. Osteopontin deficiency is associated with abnormal wound repair characterized by aberrant collagen fibrillogenesis in the heart and skin. Recent gene microarray studies found that osteopontin is abundantly expressed in both human and mouse lung fibrosis. Macrophages and T cells are known to be major sources of osteopontin. During lung fibrosis, however, osteopontin expression continues to increase when inflammation has receded, suggesting alternative sources of ostepontin during this response. In this study, we demonstrate immunoreactivity for osteopontin in lung epithelial and inflammatory cells in human usual interstitial pneumonitis and murine bleomycin-induced lung fibrosis. After treatment with bleomycin, osteopontin-null mice develop lung fibrosis characterized by dilated distal air spaces and reduced type I collagen expression compared with wild-type controls. There is also a significant decrease in levels of active transforming growth factor-β1and matrix metalloproteinase-2 in osteopontin null mice. Type III collagen expression and total collagenase activity are similar in both groups. These results demonstrate that osteopontin expression is associated with important fibrogenic signals in the lung and that the epithelium may be an important source of osteopontin during lung fibrosis.
Essential role for cathepsin D in bleomycin-induced apoptosis of alveolar epithelial cells
Authors: Li Xiaopeng , Rayford Heather , Shu Ruijie , Zhuang Jiaju , Uhal Bruce D.
Published: February 2004

Our earlier studies showed that bleomycin-induced apoptosis of type II alveolar epithelial cells (AECs) requires the autocrine synthesis and proteolytic processing of angiotensinogen into ANG II and that inhibitors of ANG-converting enzyme (ACEis) block bleomycin-induced apoptosis (Li X, Zhang H, Soledad-Conrad V, Zhuang J, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 284: L501–L507, 2003). Given the documented role of cathepsin D (CatD) in apoptosis of other cell types, we hypothesized that CatD might be the AEC enzyme responsible for the conversion of angiotensinogen into ANG I, the substrate for ACE. Primary cultures of rat type II AECs challenged with bleomycin in vitro showed upregulation and secretion of CatD enzymatic activity and immunoreactive protein but no increases in CatD mRNA. The aspartyl protease inhibitor pepstatin A, which completely blocked CatD enzymatic activity, inhibited bleomycin-induced nuclear fragmentation by 76% and reduced bleomycin-induced caspase-3 activation by 47%. Antisense oligonucleotides against CatD mRNA reduced CatD-immunoreactive protein and inhibited bleomycin-induced nuclear fragmentation by 48%. A purified fragment of angiotensinogen (F1–14) containing the CatD and ACE cleavage sites, when applied to unchallenged AEC in vitro, yielded mature ANG II peptide and induced apoptosis. The apoptosis induced by F1–14 was inhibited 96% by pepstatin A and 77% by neutralizing antibodies specific for CatD (both P < 0.001). These data indicate a critical role for CatD in bleomycin-induced apoptosis of cultured AEC and suggest that the role(s) of CatD in AEC apoptosis include the conversion of newly synthesized angiotensinogen to ANG II.
Effect of renal denervation on dynamic autoregulation of renal blood flow
Authors: DiBona Gerald F. , Sawin Linda L.
Published: February 2004

Vasoconstrictor intensities of renal sympathetic nerve stimulation elevate the renal arterial pressure threshold for steady-state stepwise autoregulation of renal blood flow. This study examined the tonic effect of basal renal sympathetic nerve activity on dynamic autoregulation of renal blood flow in rats with normal (Sprague-Dawley and Wistar-Kyoto) and increased levels of renal sympathetic nerve activity (congestive heart failure and spontaneously hypertensive rats). Steady-state values of arterial pressure and renal blood flow before and after acute renal denervation were subjected to transfer function analysis. Renal denervation increased basal renal blood flow in congestive heart failure (+35 ± 3%) and spontaneously hypertensive rats (+21 ± 3%) but not in Sprague-Dawley and Wistar-Kyoto rats. Renal denervation significantly decreased transfer function gain (i.e., improved autoregulation of renal blood flow) and increased coherence only in spontaneously hypertensive rats. Thus vasoconstrictor intensities of renal sympathetic nerve activity impaired the dynamic autoregulatory adjustments of the renal vasculature to oscillations in arterial pressure. Renal denervation increased renal blood flow variability in spontaneously hypertensive rats and congestive heart failure rats. The contribution of vasoconstrictor intensities of basal renal sympathetic nerve activity to limiting renal blood flow variability may be important in the stabilization of glomerular filtration rate.
A simplified method for HPLC determination of creatinine in mouse serum
Authors: Yuen Peter S. T. , Dunn Stephen R. , Miyaji Takehiko , Yasuda Hideo , Sharma Kumar , Star Robert A.
Published: February 2004

Mouse models are frequently used to study renal function. However, mouse serum contains chromagens that interfere with standard picric acid-based assays for serum creatinine. Several alternative methods exist for serum creatinine measurements, including assay by high-performance liquid chromatography (HPLC), but only one has been adapted to mouse serum. Creatinine was measured in serum by acetonitrile deproteinization, followed by isocratic, cation exchange HPLC. The HPLC method was compared with a standard alkaline picrate colorimetric assay, using serum from animals with low-to-moderate renal injury. Acidification of acetonitrile with HCl in the deproteinization step produced variable results, including an extra peak that interfered with integration of the creatinine peak or loss of the creatinine peak. Deproteinizing with acetonitrile alone resulted in a more reliable measurement of serum creatinine, which was validated by a series of known additions of creatinine standard. The HPLC assay was reproducible with coefficients of variation from 1.6 to 5.1%. The picric acid assay overestimated serum creatinine, when directly compared with the HPLC assay. The extent of overestimation, up to sixfold, was greatest at normal (0.1 to 0.2 mg/dl) to moderately elevated (0.5 mg/dl) serum creatinine levels. Mouse serum contains substances that interfere with standard picric acid assays for creatinine. Our new HPLC assay can accurately detect creatinine from 5 μl of mouse serum. These results support the widespread adoption of HPLC to accurately measure serum creatinine in mouse models of renal injury.
Adding fat calories to meals after exercise does not alter glucose tolerance
Authors: Fox Amanda K. , Kaufman Amy E. , Horowitz Jeffrey F.
Published: February 2004

A single session of exercise increases insulin sensitivity for hours and even days, and dietary carbohydrate ingested after exercise alters the magnitude and duration of this effect. Although increasing systemic fatty acid availability is associated with insulin resistance, it is uncertain whether increasing dietary fat availability after exercise alters the exercise-induced increase in insulin sensitivity. The purpose of this study was to determine whether adding fat calories to meals after exercise alters glucose tolerance the next day. Seven healthy men cycled 90 min at 66 ± 2% peak oxygen uptake followed by a maximum of five high-intensity intervals. During the hours after exercise, subjects ingested three meals containing either low-fat (5% energy from fat) or high-fat (45% energy from fat) foods (Low-Fat and High-Fat groups, respectively). Each diet contained the same amount of carbohydrate and protein. An oral glucose tolerance test was performed the next morning. Muscle glycogen and intramuscular triglyceride (IMTG) concentrations were measured in muscle biopsy samples obtained immediately before exercise and the next morning. The day after exercise, muscle glycogen concentration was identical in High-Fat and Low-Fat (393 ± 70 and 379 ± 38 mmol/kg dry wt). At the same time, IMTG concentration was ∼20% greater during High-Fat compared with Low-Fat (42.5 ± 3.4 and 36.3 ± 3.3 mmol/kg dry wt; P < 0.05). Despite the addition of ∼165 g of fat to meals after exercise (∼1,500 kcal) and a resultant elevation in IMTG concentration, glucose tolerance was identical in High-Fat and Low-Fat (composite index: 8.7 ± 1.0 and 8.4 ± 1.0). In summary, as long as meals ingested in the hours after exercise contain the same carbohydrate content, the addition of ∼1,500 kcal from fat to these meals did not alter muscle glycogen resynthesis or glucose tolerance the next day.
GABAB Receptor Modulation of Rapid Inhibitory and Excitatory Neurotransmission From Subfornical Organ and Other Afferents to Median Preoptic Nucleus Neurons
Authors: Kolaj Miloslav , Bai Donglin , Renaud Leo P.
Published: February 2004

Cardiovascular and behavioral responses to circulating angiotensin require intact connectivity along the upper lamina terminalis joining the subfornical organ (SFO) with the median preoptic nucleus (MnPO). Whole cell patch-clamp recordings in sagittal rat brain slice preparations revealed that 28/40 MnPO neurons responded to electrical stimulation of SFO efferents with bicuculline-sensitive GABAA receptor-mediated inhibition and glutamate-mediated postsynaptic excitation involving AMPA and N-methyl-d-aspartate (NMDA) receptor subtypes, blockable with 2,3-dioxo-6nitro-1, 2,3,4-tetrahydrobenzo [f] quinoxaline-7-sulfoamide disodium (NBQX) and d-2-amino-4-phosphonovaleric acid (d-APV), respectively. Bath applications of baclofen induced a concentration-dependent (0.3–10 μM) reduction in these SFO-evoked postsynaptic currents, attenuation of SFO-evoked paired-pulse depression, and reduction in frequency (but not amplitude) of miniature postsynaptic currents, consistent with an action at presynaptic GABAB receptors. Baclofen's effects on miniature currents lacked sensitivity to barium, ω-conotoxin GVIA, and cadmium. Acting at postsynaptic GABAB receptors, baclofen hyperpolarized a majority of MnPO neurons by increasing a G protein–coupled inwardly rectifying potassium conductance and suppressing an N-type high-voltage–activated calcium conductance. The latter contributed to reduction in action potential afterhyperpolarization and enhanced cell firing and spike frequency adaptation when tested with a depolarizing stimulus. All baclofen-induced effects were blockable with CGP52432 . CGP52432 alone had no significant effect on SFO-evoked postsynaptic current amplitudes or paired-pulse ratios, but did induce an increase in miniature inhibitory postsynaptic current (mIPSC) frequency in 2/4 cells tested, indicating that ambient levels of GABA could activate presynaptic GABAB receptors on undefined inputs. These observations indicate that MnPO neurons receive both a GABAergic and glutamatergic innervation from SFO. Both forms of rapid neurotransmission are subject to modulation via pre- and postsynaptic GABAB receptors.
Modulation of Sympathetic and Somatomotor Function by the Ventromedial Medulla
Authors: Nason Malcolm W. , Mason Peggy
Published: February 2004

The ventromedial medulla is implicated in a variety of functions including nociceptive and cardiovascular modulation and the control of thermoregulation. To determine whether single microinjections into the ventromedial medulla elicit changes in one or multiple functional systems, the GABAA receptor antagonist bicuculline was microinjected (70 nl, 5–50 ng) into the ventromedial medulla of lightly anesthetized rats, and cardiovascular, respiratory, and nociceptive measures were recorded. Bicuculline microinjection into either the midline raphe or the laterally adjacent reticular nucleus simultaneously increased interscapular brown adipose tissue temperature, heart rate, blood pressure, expired [CO2], and respiration rate and elicited shivering. Bicuculline microinjection also decreased the noxious stimulus-evoked changes in heart rate and blood pressure, decreased the frequency of heat-evoked sighs, and suppressed the cortical desynchronization evoked by noxious stimulation. Although bicuculline suppressed the motor withdrawal evoked by noxious tail heat, it enhanced the motor withdrawal evoked by noxious paw heat, evidence for specifically patterned nociceptive modulation. Saline microinjections into midline or lateral sites had no effect on any measured variable. All bicuculline microinjections, midline or lateral, evoked the same set of physiological effects, consistent with the lack of a topographical organization within the ventromedial medulla. Furthermore, as predicted by the isodendritic morphology of cells in the ventromedial medulla, midline bicuculline microinjection increased the number of c-fos immunoreactive cells in both midline raphe and lateral reticular nuclei. In summary, 70-nl microinjections into ventromedial medulla activate cells in multiple nuclei and elicit increases in sympathetic and somatomotor tone and a novel pattern of nociceptive modulation.
Development of Intrinsic Properties and Excitability of Layer 2/3 Pyramidal Neurons During a Critical Period for Sensory Maps in Rat Barrel Cortex
Authors: Maravall Miguel , Stern Edward A. , Svoboda Karel
Published: February 2004

The development of layer 2/3 sensory maps in rat barrel cortex (BC) is experience dependent with a critical period around postnatal days (PND) 10–14. The role of intrinsic response properties of neurons in this plasticity has not been investigated. Here we characterize the development of BC layer 2/3 intrinsic responses to identify possible sites of plasticity. Whole cell recordings were performed on pyramidal cells in acute BC slices from control and deprived rats, over ages spanning the critical period (PND 12, 14, and 17). Vibrissa trimming began at PND 9. Spiking behavior changed from phasic (more spike frequency adaptation) to regular (less adaptation) with age, such that the number of action potentials per stimulus increased. Changes in spiking properties were related to the strength of a slow Ca2+-dependent afterhyperpolarization. Maturation of the spiking properties of layer 2/3 pyramidal neurons coincided with the close of the critical period and was delayed by deprivation. Other measures of excitability, including I-f curves and passive membrane properties, were affected by development but unaffected by whisker deprivation.
Inhibition of Visual Discrimination During a Memory-Guided Saccade Task
Authors: Ostendorf Florian , Finke Carsten , Ploner Christoph J.
Published: February 2004

Voluntary behavior critically depends on attentional selection and short-term maintenance of perceptual information. Recent research suggests a tight coupling of both cognitive functions with visual processing being selectively enhanced by working memory representations. Here, we combined a memory-guided saccade paradigm (6-s delay) with a visual discrimination task, performed either 1,500, 2,500, or 3,500 ms after presentation of the memory cue. Contrary to what can be expected from previous studies, our results show that memory of spatial cues can transiently delay speeded discrimination of stimuli presented at remembered locations. This effect was not observed in a control experiment without memory requirements. Furthermore, delayed discrimination was dependent on the strength of actual memory representations as reflected by accuracy of memory-guided saccades. We propose an active inhibitory mechanism that counteracts facilitating effects of spatial working memory, promoting flexible orienting to novel information during maintenance of spatial memoranda for intended actions. Inhibitory delay-period activity in prefrontal cortex is a likely source for this mechanism which may be mediated by prefronto-tectal projections.
Circadian Rhythm in Inhibitory Synaptic Transmission in the Mouse Suprachiasmatic Nucleus
Authors: Itri Jason , Michel Stephan , Waschek James A. , Colwell Christopher S.
Published: February 2004

It is widely accepted that most suprachiasmatic nucleus (SCN) neurons express the neurotransmitter GABA and are likely to use this neurotransmitter to regulate excitability within the SCN. To evaluate the possibility that inhibitory synaptic transmission varies with a circadian rhythm within the mouse SCN, we used whole cell patch-clamp recording in an acute brain slice preparation to record GABA-mediated spontaneous inhibitory postsynaptic currents (sIPSCs). We found that the sIPSC frequency in the dorsal SCN (dSCN) exhibited a TTX-sensitive daily rhythm that peaked during the late day and early night in mice held in a light:dark cycle. We next evaluated whether vasoactive intestinal peptide (VIP) was responsible for the observed rhythm in IPSC frequency. Pretreatment of SCN slices with VPAC1/VPAC2- or VPAC2-specific receptor antagonists prevented the increase in sIPSC frequency in the dSCN. The rhythm in sIPSC frequency was absent in VIP/peptide histidine isoleucine (PHI)-deficient mice. Finally, we were able to detect a rhythm in the frequency of inhibitory synaptic transmission in mice held in constant darkness that was also dependent on VIP and the VPAC2 receptor. Overall, these data demonstrate that there is a circadian rhythm in GABAergic transmission in the dorsal region of the mouse SCN and that the VIP is required for expression of this rhythm.
Information Transfer Rate of Nonspiking Afferent Neurons in the Crab
Authors: DiCaprio Ralph A.
Published: February 2004

The thoracic-coxal muscle receptor organ (TCMRO) is the only proprioceptor at the thoracic-coxal joint in the crab leg. The S and T afferent neurons of the TCMRO convey signals to the CNS solely by means of graded changes in membrane potential. The rate of information transfer of these afferents was determined by measuring the signal-to-noise ratio (SΝR) of these cells after repeated stimulation of the receptor with identical sequences of random movement and applying the Shannon formula for the information capacity of a Gaussian channel. Intracellular recordings were made from the S and T afferents adjacent to the transduction site at the origin of the receptor and along the axon 5–7 mm distal to this site. These nonspiking afferents transduce receptor movement and transmit this information with extremely high fidelity. The SNR of both neurons near the transduction site was >1000 over most of the 200 Hz stimulation bandwidth, and the mean information transfer rate was ∼2,500 bits/s. When calculated over a wider bandwidth of 500 Hz, the information rate was >4,600 bits/s. The effect of axonal cable properties on the information rate was evaluated by determining the SNR from membrane potential recordings made 5–7 mm distal to the transduction region. The major effect of graded transmission along the axon was attenuation and low-pass filtering of the sensory signal. The consequent reduction in signal power and bandwidth decreased the information transfer by ∼10–15% over 200 Hz and ∼30% over a 500 Hz bandwidth.
Signaling in Leukocyte Transendothelial Migration
Authors: van Buul Jaap D. , Hordijk Peter L.
Published: February 2004

Under a variety of (patho) physiological conditions, leukocytes will leave the bloodstream by crossing the endothelial monolayer that lines the vessels and migrate into the underlying tissues. It is now clear that the process of extravasation involves a range of adhesion molecules on both leukocytes and endothelial cells, as well as extensive intracellular signaling that drives adhesion and chemotaxis on the one hand and controls a transient modulation of endothelial integrity on the other. We review here the current knowledge of the intracellular signaling pathways that are activated in the context of transendothelial migration in leukocytes and in endothelial cells. In leukocytes, polarization of receptors and of the signaling machinery is of key importance to drive adhesion and directional migration. Subsequent adhesion-induced signaling in endothelial cells, mediated by Rho-like GTPases and reactive oxygen species, induces a transient and focal loss of endothelial cell–cell adhesion to allow transmigration of the leukocyte. This review underscores the notion that we have likely just scratched the surface in revealing the complexity of the signaling that controls leukocyte transendothelial migration.
Randomized Study to Evaluate Sirolimus-Eluting Stents Implanted at Coronary Bifurcation Lesions
Authors: Colombo Antonio , Moses Jeffrey W. , Morice Marie Claude , Ludwig Josef , Holmes David R. , Spanos Vassilis , Louvard Yves , Desmedt Benny , Di Mario Carlo , Leon Martin B.
Published: February 2004

Background—

A sirolimus-eluting stent (Cypher, Cordis Corp) has been reported to markedly decrease restenosis in selected lesions; higher-risk lesions, including coronary bifurcations, have not been studied.

Methods and Results—

This prospective study evaluated the safety and efficacy of sirolimus-eluting stents for treatment of coronary bifurcation lesions. Patients were randomly assigned to either stenting of both branches (group A) or stenting of the main branch with provisional stenting of the side branch (SB) (group B). Eighty-five patients (86 lesions) were enrolled. There was 1 case of unsuccessful delivery of any device at the bifurcation site. Given the high crossover, more lesions were treated with 2 stents (n=63) than with stent/balloon (n=22). Clinical follow-up at 6 months was completed in all patients and angiographic follow-up in 53 patients in group A (85.5%) and 21 in group B (95.4%). One patient died suddenly 4.5 months after the procedure. There were 3 cases of stent thrombosis (3.5%). The total restenosis rate at 6 months was 25.7%, and it was not significantly different between the double-stenting (28.0%) and the provisional SB-stenting (18.7%) groups. Fourteen of the restenosis cases occurred at the ostium of the SB and were focal. Target lesion revascularization was performed in 7 cases; target vessel failure occurred in 15 cases (17.6%).

Conclusions—

These results are an improvement compared with historical controls using bare metal stents. Restenosis at the SB remains a problem. At this time, no statement can be made regarding the most appropriate technique to use when treating bifurcations with the Cypher stent.
Endothelin-A Receptor Antagonism Reduces Blood Pressure and Increases Renal Blood Flow in Hypertensive Patients With Chronic Renal Failure
Authors: Goddard Jane , Johnston Neil R. , Hand Malcolm F. , Cumming Allan D. , Rabelink Ton J. , Rankin Andrew J. , Webb David J.
Published: February 2004

Background—

Endothelin (ET) is implicated in the pathophysiology of chronic renal failure (CRF). We therefore studied the systemic and renal hemodynamic effects of ET receptor antagonists in CRF and examined differences between selective ETA, selective ETB, and combined ETA/B receptor blockade.

Methods and Results—

We conducted a randomized, placebo-controlled, double-blind, 4-way crossover study comparing selective ET receptor antagonists BQ-123 (ETA) and BQ-788 (ETB), given alone and in combination, in acute studies in 8 hypertensive CRF patients and 8 matched healthy controls. BQ-123, alone and in combination with BQ-788, reduced blood pressure in CRF, particularly with BQ-123 alone (mean arterial pressure: controls −4±2%, CRF −13±2%,
P
<0.01 versus placebo). In CRF, in the face of this fall in blood pressure, BQ-123 substantially increased renal blood flow (38.8±23.9%,
P
<0.01 versus placebo) and reduced renal vascular resistance (−44.5±11.3%,
P
<0.01 versus placebo) when given alone but not when combined with BQ-788. These changes were accompanied by a reduction in effective filtration fraction. BQ-123, alone or in combination with BQ-788, had minimal effects on the renal circulation in healthy controls, and BQ-788 alone produced both systemic and renal vasoconstriction in CRF and healthy controls.

Conclusions—

ETA receptor antagonism was highly effective in lowering blood pressure in CRF patients currently treated for hypertension. In addition, there were effects consistent with a renoprotective action. However, because the ETB receptor appears to play a key role in the maintenance of tonic renal vasodilation, combined ETA/B receptor antagonism, although it lowered blood pressure, did not confer these renal benefits.
Effects of Glucagon-Like Peptide-1 in Patients With Acute Myocardial Infarction and Left Ventricular Dysfunction After Successful Reperfusion
Authors: Nikolaidis Lazaros A. , Mankad Sunil , Sokos George G. , Miske Glen , Shah Ankur , Elahi Dariush , Shannon Richard P.
Published: February 2004

Background—

Glucose-insulin-potassium infusions are beneficial in uncomplicated patients with acute myocardial infarction (AMI) but are of unproven efficacy in AMI with left ventricular (LV) dysfunction because of volume requirements associated with glucose infusion. Glucagon-like peptide-1 (GLP-1) is a naturally occurring incretin with both insulinotropic and insulinomimetic properties that stimulate glucose uptake without the requirements for concomitant glucose infusion.

Methods and Results—

We investigated the safety and efficacy of a 72-hour infusion of GLP-1 (1.5 pmol/kg per minute) added to background therapy in 10 patients with AMI and LV ejection fraction (EF) <40% after successful primary angioplasty compared with 11 control patients. Echocardiograms were obtained after reperfusion and after the completion of the GLP-1 infusion. Baseline demographics and background therapy were similar, and both groups had severe LV dysfunction at baseline (LVEF=29±2%). GLP-1 significantly improved LVEF (from 29±2% to 39±2%,
P
<0.01), global wall motion score indexes (1.94±0.11→1.63±0.09,
P
<0.01), and regional wall motion score indexes (2.53±0.08→2.02±0.11,
P
<0.01) compared with control subjects. The benefits of GLP-1 were independent of AMI location or history of diabetes. GLP-1 was well tolerated, with only transient gastrointestinal effects.

Conclusions—

When added to standard therapy, GLP-1 infusion improved regional and global LV function in patients with AMI and severe systolic dysfunction after successful primary angioplasty.