Extracellular pressure stimulates macrophage phagocytosis by inhibiting a pathway involving FAK and ERK

We hypothesized that changes in extracellular pressure during inflammation or infection regulate macrophage phagocytosis through modulating the focal adhesion kinase (FAK)-ERK pathway. Undifferentiated (monocyte-like) or PMA-differentiated (macrophage-like) THP-1 cells were incubated at 37°C with serum-opsonized latex beads under ambient or 20-mmHg increased pressure. Pressure did not affect monocyte phagocytosis but significantly increased macrophage phagocytosis (29.9 ± 1.8 vs. 42.0 ± 1.6%, n = 9, P < 0.001). THP-1 macrophages constitutively expressed activated FAK, ERK, and Src. Exposure of macrophages to pressure decreased ERK and FAK-Y397 phosphorylation (77.6 ± 7.9%, n = 7, P < 0.05) but did not alter FAK-Y576 or Src phosphorylation. FAK small interfering RNA (SiRNA) reduced FAK expression by >75% and the basal amount of phosphorylated FAK by 25% and significantly increased basal macrophage phagocytosis (P < 0.05). Pressure inhibited FAK-Y397 phosphorylation in mock-transfected or scrambled SiRNA-transfected macrophages, but phosphorylated FAK was not significantly reduced further by pressure in cells transfected with FAK SiRNA. Pressure increased phagocytosis in all three groups. However, FAK-SiRNA-transfected cells exhibited only 40% of the pressure effect on phagocytosis observed in scrambled SiRNA-transfected cells so that phagocytosis inversely paralleled FAK activation. PD-98059 (50 µM), an ERK activation inhibitor, increased basal phagocytosis (26.9 ± 1.8 vs. 31.7 ± 1.1%, n = 15, P < 0.05), but pressure did not further increase phagocytosis in PD-98059-treated cells. Pressure also inhibited ERK activation after mock transfection or transfection with scrambled SiRNA, but transfection of FAK SiRNA abolished ERK inhibition by pressure. Pressure did not increase phagocytosis in MonoMac-1 cells that do not express FAK. Increased extracellular pressure during infection or inflammation enhances macrophage phagocytosis by inhibiting FAK and, consequently, decreasing ERK activation. force; inflammation; infection; leukocyte; mechanotransduction; signal transduction     

Selective knockdown of AT1 receptors by RNA interference inhibits Val5-ANG II endocytosis and NHE-3 expression in immortalized rabbit proximal tubule cells

Receptor-mediated endocytosis of extracellular ANG II has been suggested to play an important role in the regulation of proximal tubule cell (PTC) function. Using immortalized rabbit PTCs as an in vitro cell culture model, we tested the hypothesis that extracellular ANG II is taken up by PTCs through angiotensin type 1 receptor (AT₁; or AT_1a) receptor-mediated endocytosis and that inhibition of ANG II endocytosis using a selective AT₁ receptor small-interfering RNA (siRNA; AT₁R siRNA) or endocytotic inhibitors exerts a physiological effect on total and apical sodium and hydrogen exchanger isoform 3 (NHE-3) protein abundance. Western blots and live cell imaging with FITC-labeled ANG II confirmed that transfection of PTCs with a human specific AT₁R siRNA for 48 h selectively knocked down AT₁ receptor protein by 76 ± 5% (P < 0.01), whereas transfection with a scrambled siRNA had little effect. In nontransfected PTCs, exposure to extracellular ANG II (1 nM) for 60 min at 37°C increased intracellular ANG II accumulation by 67% (control: 566 ± 55 vs. ANG II: 943 ± 160 pg/mg protein, P < 0.05) and induced mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) 1/2 phosphorylation (163 ± 15% of control, P < 0.01). AT₁R siRNA reduced ANG II endocytosis to a level similar to losartan, which blocks cell surface AT₁ receptors (557 ± 37 pg/mg protein, P < 0.05 vs. ANG II), or to colchicine, which disrupts cytoskeleton microtubules (613 ± 12 pg/mg protein, P < 0.05 vs. ANG II). AT₁R siRNA, losartan, and colchicine all attenuated ANG II-induced ERK1/2 activation and total cell lysate and apical membrane NHE-3 abundance. The scrambled siRNA had no effect on ANG II endocytosis, ERK1/2 activation, or NHE-3 expression. These results suggest that AT₁ receptor-mediated endocytosis of extracellular ANG II may regulate proximal tubule sodium transport by increasing total and apical NHE-3 proteins. extracellular signal-regulated kinase 1/2; kidney; sodium transport; receptor internalization; ribonucleic acid interference     

Changes in agonist-antagonist EMG, muscle CSA, and force during strength training in middle-aged and older people

Effects of 6 mo of heavy-resistance trainingcombined with explosive exercises on neural activation of the agonistand antagonist leg extensors, muscle cross-sectional area (CSA) of thequadriceps femoris, as well as maximal and explosive strength wereexamined in 10 middle-aged men (M40; 42 ± 2 yr), 11 middle-agedwomen (W40; 39 ± 3 yr), 11 elderly men (M70; 72 ± 3 yr) and 10 elderly women (W70; 67 ± 3 yr). Maximal andexplosive strength remained unaltered during a 1-mo control period withno strength training. After the 6 mo of training, maximal isometric anddynamic leg-extension strength increased by 36 ± 4 and 22 ± 2%(P < 0.001) in M40, by 36 ± 3 and 21 ± 3% (P < 0.001) in M70,by 66 ± 9 and 34 ± 4% (P < 0.001) in W40, and by 57 ± 10 and 30 ± 3%(P < 0.001) in W70, respectively.All groups showed large increases (P < 0.05-0.001) in the maximum integrated EMGs (iEMGs) of theagonist vastus lateralis and medialis. Significant(P < 0.05-0.001) increasesoccurred in the maximal rate of isometric force productionand in a squat jump that were accompanied with increased(P < 0.05-0.01) iEMGs of theleg extensors. The iEMG of the antagonist biceps femoris muscle duringthe maximal isometric leg extension decreased in both M70 (from 24 ± 6 to 21 ± 6%; P < 0.05)and in W70 (from 31 ± 9 to 24 ± 4%;P < 0.05) to the same level asrecorded for M40 and W40. The CSA of the quadriceps femoris increasedin M40 by 5% (P < 0.05), in W40 by9% (P < 0.01), in W70 by 6%(P < 0.05), and in M70 by 2% (notsignificant). Great training-induced gains in maximal and explosivestrength in both middle-aged and elderly subjects were accompanied bylarge increases in the voluntary activation of the agonists, withsignificant reductions in the antagonist coactivation in the elderlysubjects. Because the enlargements in the muscle CSAs in bothmiddle-aged and elderly subjects were much smaller in magnitude, neuraladaptations seem to play a greater role in explaining strength andpower gains during the present strength-training protocol.

     

Shortening velocity and myosin heavy- and light-chain isoform mRNA in rabbit arterial smooth muscle cells

In smooth muscle cells (SMCs)isolated from rabbit carotid, femoral, and saphenous arteries, relativemyosin isoform mRNA levels were measured in RT-PCR to test forcorrelations between myosin isoform expression and unloaded shorteningvelocity. Unloaded shortening velocity and percent smooth muscle myosinheavy chain 2 (SM2) and myosin light chain 17b(MLC_17b) mRNA levels were not significantly different insingle SMCs isolated from the luminal and adluminal regions of thecarotid media. Saphenous artery SMCs shortened significantly faster(P < 0.05) than femoral SMCs and had more SM2 mRNA(P < 0.05) than carotid SMCs and lessMLC_17b mRNA (P < 0.001) and higher tissuelevels of SMB mRNA (P < 0.05) than carotid and femoralSMCs. No correlations were found between percent SM2 and percentMLC_17b mRNA levels and unloaded shortening velocity in SMCsfrom these arteries. We have previously shown that myosin heavy chain(MHC) SM1/SM2 and SMA/SMB and MLC_17a/MLC_17b isoform mRNA levels correlate with protein expression for these isoforms in rabbit smooth muscle tissues. Thus we interpret these results to suggest that 1) SMC myosin isoform expression andunloaded shortening velocity do not vary with distance from the lumenof the carotid artery but do vary in arteries located longitudinally within the arterial tree, 2) MHC SM1/SM2 and/orMLC_17a/MLC_17b isoform expression does notcorrelate with unloaded shortening velocity, and 3)intracellular expression of the MHC SM1/SM2 and MLC_17a/MLC_17b isoforms is not coregulated.

     

Mechanical strain induces a persistent upregulation of syndecan-1 expression in smooth muscle cells

Syndecan-1 belongs to a family of transmembrane proteoglycans, acts as a coreceptor for growth factor binding, as well as cell-matrix and cell-cell interactions, and is induced in smooth muscle cells (SMCs) following balloon catheter injury. In this report, we investigated syndecan-1 expression in SMCs in response to several distinct biomechanical force profiles and the related syndecan shedding response. Syndecan-1 mRNA expression increased in response to 5% and 10% cyclic strain (24 h: 206 +/- 40% and 278 +/- 33%, respectively, P < 0.05) when compared to unstrained controls. When subjected to 10% cyclic strain for periods of up to 48 h, syndecan-1 mRNA levels remained elevated at 294 +/- 31%. Notably, the SMC mechanosensor mechanism remained responsive after an initial 24 h "preconditioning" period, as evident by a fivefold increase in syndecan-1 gene expression following a change in cyclic stress from 10% to 20% (48 h: 516 +/- 55%, P < 0.05). Of note, similar behavior was not observed in an analysis of syndecan-2 mRNA levels. Commensurate with mRNA responses, mechanical stress induced an increase in cell-associated syndecan-1 protein levels with an associated increase in protein shedding. Given the varied functions of syndecan-1, stress-induced effects on SMC syndecan-1 expression and shedding may represent an additional component of the pro-inflammatory, growth-stimulating pathways that are activated in response to changes in the mechanical microenvironment of the vascular wall. Syndecan-1 expression is uniquely influenced by changes in the phase and magnitude of the local stress field.     

Effect of acute head-down tilt on skeletal muscle cross-sectional area and proton transverse relaxation time

Conley, Michael S., Jeanne M. Foley, Lori L. Ploutz-Snyder,Ronald A. Meyer, and Gary A. Dudley. Effect of acute head-down tilt on skeletal muscle cross-sectional area and proton transverse relaxation time. J. Appl. Physiol.81(4): 1572-1577, 1996.This study investigated changes inskeletal muscle cross-sectional area (CSA) evoked by fluid shifts thataccompany short-term 6° head-down tilt (HDT) or horizontal bedrest, the time course of the resolution of these changes afterresumption of upright posture, and the effect of altered muscle CSA, inthe absence of increased contractile activity, on proton transverserelaxation time (T₂). Averagemuscle CSA and T₂ were determinedby standard spin-echo magnetic resonance imaging. Analyses wereperformed on contiguous transaxial images of the neck and calf. After aday of normal activity, 24 h of HDT increased neck muscle CSA 19 ± 4 (SE)% (P < 0.05) whilecalf muscle CSA decreased 14 ± 3%(P < 0.05). The horizontal posture(12 h) induced about one-half of these responses: an 11 ± 2%(P < 0.05) increase in neck muscleCSA and an 8 ± 2% decrease (P < 0.05) in the calf. Within 2 h after resumption of upright posture, neckand calf muscle CSA returned to within 0.5% (P > 0.05) of the values assessedafter a day of normal activity, with most of the change occurringwithin the first 30 min. No further change in muscle CSA was observedthrough 6 h of upright posture. Despite these large alterations inmuscle CSA, T₂ was not altered bymore than 1.1 ± 0.6% (P > 0.05)and did not relate to muscle size. These results suggest that posturalmanipulations and subsequent fluid shifts modeling microgravity elicitmarked changes in muscle size. Because these responses were notassociated with alterations in muscleT₂, it does not appear that simple movement of water into muscle can explain the contrast shift observed after exercise.

     

Transcapillary escape rate of albumin in humans during exercise-induced hypervolemia

Haskell, Andrew, Ethan R. Nadel, Nina S. Stachenfeld, KeiNagashima, and Gary W. Mack. Transcapillary escape rate of albuminin humans during exercise-induced hypervolemia. J. Appl. Physiol. 83(2): 407-413, 1997.To test thehypotheses that plasma volume (PV) expansion 24 h after intenseexercise is associated with reduced transcapillary escape rate ofalbumin (TER_alb) and that localchanges in transcapillary forces in the previously active tissues favorretention of protein in the vascular space, we measured PV,TER_alb, plasma colloid osmoticpressure (COP_p), interstitialfluid hydrostatic pressure (Pi), and colloid osmotic pressure in legmuscle and skin and capillary filtration coefficient (CFC) in the armand leg in seven men and women before and 24 h after intense uprightcycle ergometer exercise. Exercise expanded PV by 6.4% at 24 h (43.9 ± 0.8 to 46.8 ± 1.2 ml/kg, P < 0.05) and decreased total protein concentration (6.5 ± 0.1 to6.3 ± 0.1 g/dl, P < 0.05) andCOP_p (26.1 ± 0.8 to 24.3 ± 0.9 mmHg, P < 0.05), although plasmaalbumin concentration was unchanged. TER_alb tended to decline (8.4 ± 0.5 to 6.5 ± 0.7%/h, P = 0.11) and was correlated with the increase in PV(r = 0.69,P < 0.05). CFC increased in the leg(3.2 ± 0.2 to 4.3 ± 0.5 µl · 100 g¹ · min¹ · mmHg¹,P < 0.05), and Pi showed a trend toincrease in the leg muscle (2.8 ± 0.7 to 3.8 ± 0.3 mmHg, P = 0.08). These datademonstrate that TER_alb isassociated with PV regulation and that local transcapillary forcesin the leg muscle may favor retention of albumin in the vascular spaceafter exercise.

     

Effects of four different single exercise sessions on lipids, lipoproteins, and lipoprotein lipase

The purpose ofthis study was to determine the threshold of exercise energyexpenditure necessary to change blood lipid and lipoproteinconcentrations and lipoprotein lipase activity (LPLA) in healthy,trained men. On different days, 11 men (age, 26.7 ± 6.1 yr; bodyfat, 11.0 ± 1.5%) completed four separate, randomly assigned,submaximal treadmill sessions at 70% maximalO₂ consumption. During eachsession 800, 1,100, 1,300, or 1,500 kcal were expended. Compared withimmediately before exercise, high-density lipoprotein cholesterol(HDL-C) concentration was significantly elevated 24 h after exercise(P < 0.05) in the 1,100-, 1,300-, and 1,500-kcal sessions. HDL-C concentration was also elevated(P < 0.05) immediately after and 48 h after exercise in the 1,500-kcal session. Compared with values 24 hbefore exercise, LPLA wassignificantly greater (P < 0.05) 24 h after exercise in the 1,100-, 1,300-, and 1,500-kcal sessions andremained elevated 48 h after exercise in the 1,500-kcal session. Thesedata indicate that, in healthy, trained men, 1,100 kcal of energyexpenditure are necessary to elicit increased HDL-C concentrations.These HDL-C changes coincided with increased LPLA.

     

Brain natriuretic peptide inhibits hypoxic pulmonary hypertension in rats

Brainnatriuretic peptide (BNP) is a pulmonary vasodilator that is elevatedin the right heart and plasma of hypoxia-adapted rats. To test thehypothesis that BNP protects against hypoxic pulmonary hypertension, wemeasured right ventricular systolic pressure (RVSP), right ventricle(RV) weight-to-body weight (BW) ratio (RV/BW), and percentmuscularization of peripheral pulmonary vessels (%MPPV) in rats givenan intravenous infusion of BNP, atrial natriuretic peptide (ANP), orsaline alone after 2 wk of normoxia or hypobaric hypoxia (0.5 atm).Hypoxia-adapted rats had higher hematocrits, RVSP, RV/BW, and %MPPVthan did normoxic controls. Under normoxic conditions, BNP infusion(0.2 and 1.4 µg/h) increased plasma BNP but had no effect on RVSP,RV/BW, or %MPPV. Under hypoxic conditions, low-rate BNP infusion (0.2 µg/h) had no effect on plasma BNP or on severity of pulmonaryhypertension. However, high-rate BNP infusion (1.4 µg/h) increasedplasma BNP (69 ± 8 vs. 35 ± 4 pg/ml, P < 0.05),lowered RV/BW (0.87 ± 0.05 vs. 1.02 ± 0.04, P < 0.05), and decreased %MPPV (60 vs. 74%,P < 0.05). There was also a trend towardlower RVSP (55 ± 3 vs. 64 ± 2, P = not significant).Infusion of ANP at 1.4 µg/h increased plasma ANP in hypoxic rats (759 ± 153 vs. 393 ± 54 pg/ml, P < 0.05) but had noeffect on RVSP, RV/BW, or %MPPV. We conclude that BNP may regulatepulmonary vascular responses to hypoxia and, at the doses used in thisstudy, is more effective than ANP at blunting pulmonary hypertensionduring the first 2 wk of hypoxia.

     

Functional characterization of two S-nitroso-L-cysteine transporters, which mediate movement of NO equivalents into vascular cells

In myogenic C₂C₁₂ cells, 5 mM creatine increased the incorporation of labeled [³⁵S]methionine into sarcoplasmic (+20%, P < 0.05) and myofibrillar proteins (+50%, P < 0.01). Creatine also promoted the fusion of myoblasts assessed by an increased number of nuclei incorporated within myotubes (+40%, P < 0.001). Expression of myosin heavy chain type II (+1,300%, P < 0.001), troponin T (+65%, P < 0.01), and titin (+40%, P < 0.05) was enhanced by creatine. Mannitol, taurine, and -alanine did not mimic the effect of creatine, ruling out an osmolarity-dependent mechanism. The addition of rapamycin, the inhibitor of mammalian target of rapamycin/70-kDa ribosomal S6 protein kinase (mTOR/p70^s6k) pathway, and SB 202190, the inhibitor of p38, completely blocked differentiation in control cells, and creatine did not reverse this inhibition, suggesting that the mTOR/p70^s6k and p38 pathways could be potentially involved in the effect induced by creatine on differentiation. Creatine upregulated phosphorylation of protein kinase B (Akt/PKB; +60%, P < 0.001), glycogen synthase kinase-3 (+70%, P < 0.001), and p70^s6k (+50%, P < 0.001). Creatine also affected the phosphorylation state of p38 (–50% at 24 h and +70% at 96 h, P < 0.05) as well as the nuclear content of its downstream targets myocyte enhancer factor-2 (–55% at 48 h and +170% at 96 h, P < 0.05) and MyoD (+60%, P < 0.01). In conclusion, this study points out the involvement of the p38 and the Akt/PKB-p70^s6k pathways in the enhanced differentiation induced by creatine in C₂C₁₂ cells. protein synthesis; insulin-like growth factor; mitogen-activated protein kinase; extracellular signal-regulated kinase 1/2; 70-kDa ribosomal S6 protein kinase     

Systemic and pulmonary hemodynamic responses to normal and obstructed breathing during sleep

Schneider, H., C. D. Schaub, K. A. Andreoni, A. R. Schwartz,R. L. Smith, J. L. Robotham, and C. P. O'Donnell. Systemic andpulmonary hemodynamic responses to normal and obstructed breathing during sleep. J. Appl. Physiol. 83(5):1671-1680, 1997.We examined the hemodynamic responses to normalbreathing and induced upper airway obstructions during sleep in acanine model of obstructive sleep apnea. During normal breathing,cardiac output decreased (12.9 ± 3.5%,P < 0.025) from wakefulness tonon-rapid-eye-movement sleep (NREM) but did not change from NREM torapid-eye-movement (REM) sleep. There was a decrease(P < 0.05) in systemic (7.2 ± 2.1 mmHg) and pulmonary (2.0 ± 0.6 mmHg) arterial pressures fromwakefulness to NREM sleep. In contrast, systemic (8.1 ± 1.0 mmHg,P < 0.025), but not pulmonary,arterial pressures decreased from NREM to REM sleep. During repetitiveairway obstructions (56.0 ± 4.7 events/h) in NREM sleep, cardiacoutput (17.9 ± 3.1%) and heart rate (16.2 ± 2.5%) increased(P < 0.05), without a change instroke volume, compared with normal breathing during NREM sleep. Duringsingle obstructive events, left (7.8 ± 3.0%,P < 0.05) and right (7.1 ± 0.7%, P < 0.01)ventricular outputs decreased during the apneic period. However, left(20.7 ± 1.6%, P < 0.01) andright (24.0 ± 4.2%, P < 0.05)ventricular outputs increased in the postapneic period because of anincrease in heart rate. Thus 1) thesystemic, but not the pulmonary, circulation vasodilates during REMsleep with normal breathing; 2)heart rate, rather than stroke volume, is the dominant factormodulating ventricular output in response to apnea; and3) left and right ventricular outputs oscillate markedly and in phase throughout the apnea cycle.

     

Tissue factor activity is increased in human endothelial cells cultured under elevated static pressure

We tested the hypothesis that elevated blood pressure, a knownstimulus for vascular remodeling and an independent risk factor for thedevelopment of atherosclerotic disease, can modulate basal andcytokine-induced tissue factor (TF; CD 142) expression in culturedhuman endothelial cells (EC). Using a chromogenic enzymatic assay, wemeasured basal and tumor necrosis factor- (TNF-; 10 ng/ml, 5 h)-induced TF activities in human aortic EC (HAEC) and vena cava EC(HVCEC) cultured at atmospheric pressure and at 170 mmHg imposedpressure for up to 48 h. Basal TF activities were 22 ± 10 U/mgprotein for HAEC and 14 ± 9 U/mg protein for HVCEC and wereupregulated in both cell types >10-fold by TNF-. Exposure topressure for 5 h induced additional elevation of basal TF activity by47 ± 16% (P < 0.05, n = 6) for HAEC and 17 ± 5%(P < 0.05, n = 3) for HVCEC. Pressurization alsoenhanced TF activity in TNF--treated cells from 240 ± 28 to 319 ± 32 U/mg protein in HAEC (P < 0.05, n = 4) and from 148 ± 25 to179 ± 0.8 U/mg protein (P < 0.05, n = 3) in HVCEC. Cytokinestimulation caused an ~100-fold increase in steady-state TF mRNAlevels in HAEC, whereas pressurization did not alter either TF mRNA orcell surface antigen expression, as determined by quantitative RT-PCRmethodology and ELISA. Elevated pressure, however, modulated the ECplasma membrane organization and/or permeability as inferred from theincreased cellular uptake of the fluorescent amphipathic dyemerocyanine 540 (33 ± 7%, P < 0.05). Our data suggest that elevated static pressure modulates thehemostatic potential of vascular cells by modifying the molecular organization of the plasma membrane.

     

Effect of exercise timing on postprandial lipemia and HDL cholesterol subfractions

The purpose ofthe study was to examine the effect of exercise timing on postprandiallipemia responses. Subjects were 21 recreationally trained men (ages 27 ± 1.7 yr). Each subject performed four trials:1) Control (fat meal only),2) Post (exercise 1 h after a fat meal), 3) 1 h-Pre(exercise 1 h before a fat meal), and4) 12 h-Pre (exercise 12 h before afat meal). In each trial, subjects had a standard fat meal to inducepostprandial hypertriglyceridemia. Blood samples were taken at 0 h(immediately before the fat meal) and at 2, 4, 6, 8, and 24 h after themeal. In the exercise trials, each subject exercised at 60% of maximalO₂ consumption for 1 h. Theresults indicated that triglyceride area under the curve scores inpremeal-exercise trials were lower (P < 0.05) than those in Post and Control. At 24 h, total high-densitylipoprotein (HDL)-cholesterol in the premeal-exercise trials was higher(P < 0.05) than that at 0 h, whereastotal HDL-cholesterol was not changed in Control and Post. At 24 h, HDLsubtype 2-cholesterol was higher (P < 0.05) in the premeal-exercise trials than in Control, which did not differ from Post. These results suggest that exercising before a fatmeal may have a beneficial effect on the triglyceride response and HDLmetabolism, which may blunt atherosclerotic process induced by the fatmeal.

     

Benzo[a]pyrene-induced cell cycle progression is through ERKs/cyclin D1 pathway and requires the activation of JNKs and p38 mapk in human diploid lung fibroblasts

Treatment of cells with carcinogen Benzo[a]pyrene (B[a]P) allows cells to evade G1 arrest and induces cells abnormal proliferation. However, the mechanisms of its action at cellular level are not well understood. To address this question, normal human embryo lung diploid fibroblasts (HELF) were selected in the present study. We found that exposure of cells with 2.5 μM of B[a]P for 24 h resulted in a decrease of G1 population by 11.9% (P < 0.05) and a increase of S population by 17.2% (P < 0.05). Treatment of cells with B[a]P also caused dose-related activation of MAPK and induction of cyclin D1 protein expression, whereas the CDK4 protein levels were not significantly affected by B[a]P. Overexpression of cyclin D1 protein stimulated by B[a]P was significantly inhibited by 50 μM AG126 (an inhibitor of ERK1/2), but not by 25 μM SP600125 (an inhibitor of JNK1/2) or 5 μM SB203580 (an inhibitor of p38 mapk), suggesting that B[a]P-induced cyclin D1 expression was only regulated by ERK1/2 pathway. However, AG126, SP600125 or SB203580 led to cell cycle significantly arrested in G1 phase, indicating that ERK1/2, JNK1/2 and p38 mapk pathways are all required for B[a]P-induced G1/S transition. In addition, HELF cells transfecting with antisense cyclin D1 cDNA or antisense CDK4 cDNA showed significantly G1 arrest after B[a]P stimulation. These results suggested that B[a]P exposure accelerated the G1→S transition by activation of MAPK signaling pathways. Cyclin D1 and CDK4 are rate-limiting regulators of the G1→S transition and expression of cyclin D1 is predominantly regulated by ERK1/2 pathway in HELF cells.     

Changes in cellular composition of kidney collecting duct cells in rats with lithium-induced NDI

Lithium treatment for 4 wk caused severe polyuria, dramatic downregulation in aquaporin-2 (AQP-2) expression, and marked decrease in AQP-2 immunoreactivity with the appearance of a large number of cells without AQP-2 labeling in the collecting ducts after lithium treatment. Surprisingly, this was not all due to an increase in AQP-2-negative principal cells, because double immunolabeling revealed that the majority of the AQP-2-negative cells displayed [H⁺]ATPase labeling, which identified them as intercalated cells. Moreover, multiple [H⁺]ATPase-labeled cells were adjacent, which was never seen in control rats. Quantitation confirmed a significant decrease in the fraction of collecting duct cells that exhibited detectable AQP-2 labeling compared with control rats: in cortical collecting ducts, 40 ± 3.4 vs. 62 ± 1.8% of controls (P < 0.05; n = 4) and in inner medullary collecting ducts, 58 ± 1.6 vs. 81 ± 1.3% of controls (P < 0.05; n = 4). In parallel, a significant increase in the fraction of intercalated ([H⁺]ATPase-positive) cells was shown. Urine output, whole kidney AQP-2 expression, cellular organization, and the fractions of principal and intercalated cells in cortex and inner medulla returned to control levels after 4 wk on a lithium-free diet following 4 wk on a lithium-containing diet. In conclusion, lithium treatment not only decreased AQP-2 expression, but dramatically and reversibly reduced the fraction of principal cells and altered the cellular organization in collecting ducts. These effects are likely to be important in lithium-induced nephrogenic diabetes insipidus. nephrogenic diabetes insipidus; aquaporin; exchanger     

Leukocyte adhesion molecule expression during intense resistance exercise

We hypothesized that expression of L-selectinand very late antigen-4 (VLA-4) integrin adhesionmolecules would influence cell type-specific redistribution duringexercise. Women subjects performed six sets of 10-repetition maximumsquats. L-selectin and VLA-4 integrin were measured by using flowcytometry pre- and postexercise on peripheral blood neutrophils andlymphocytes (n = 29 subjects) andlymphocyte subsets (n = 70 subjects),respectively. Neutrophil concentration increased 41.8%(P < 0.001), whereas the percentexpressing L-selectin was unchanged (79%). Lymphocyte concentrationincreased 61.8% (P < 0.001). Thepercent of T cells expressing L-selectin decreased from 73.5 ± 8.9 to 68.2 ± 11.4% (P < 0.001); the combined population of natural killer and B cells expressing L-selectin decreased from 80.4 ± 22.5 to 62.7 ± 25.8% (P < 0.001).VLA-4 integrin was expressed by nearly all lymphocytes both pre- andpostexercise. The proportional decrease in L-selectin positive cellscould have resulted from 1) sheddingof L-selectin, 2) selective entry ofL-selectin-negative subsets, or 3)selective removal of L-selectin-positive subsets.

     

Muscle glycogen accumulation after endurance exercise in trained and untrained individuals

Hickner, R. C., J. S. Fisher, P. A. Hansen, S. B. Racette,C. M. Mier, M. J. Turner, and J. O. Holloszy. Muscle glycogen accumulation after endurance exercise in trained and untrained individuals. J. Appl. Physiol. 83(3):897-903, 1997.Muscle glycogen accumulation was determined in sixtrained cyclists (Trn) and six untrained subjects (UT) at 6 and either48 or 72 h after 2 h of cycling exercise at ~75% peakO₂ uptake(O_{2 peak}), which terminated with five 1-min sprints. Subjects ate 10 gcarbohydrate · kg¹ · day¹for 48-72 h postexercise. Muscle glycogen accumulation averaged 71 ± 9 (SE) mmol/kg (Trn) and 31 ± 9 mmol/kg (UT) during the first 6 h postexercise (P < 0.01) and 79 ± 22 mmol/kg (Trn) and 60 ± 9 mmol/kg (UT) between 6 and 48 or 72 h postexercise (not significant). Muscle glycogenconcentration was 164 ± 21 mmol/kg (Trn) and 99 ± 16 mmol/kg(UT) 48-72 h postexercise (P < 0.05). Muscle GLUT-4 content immediately postexercise was threefoldhigher in Trn than in UT (P < 0.05)and correlated with glycogen accumulation rates (r = 0.66, P < 0.05). Glycogen synthase in theactive I form was 2.5 ± 0.5, 3.3 ± 0.5, and 1.0 ± 0.3 µmol · g¹ · min¹in Trn at 0, 6, and 48 or 72 h postexercise, respectively;corresponding values were 1.2 ± 0.3, 2.7 ± 0.5, and 1.6 ± 0.3 µmol · g¹ · min¹in UT (P < 0.05 at 0 h). Plasmainsulin and plasma C-peptide area under the curve were lower in Trnthan in UT over the first 6 h postexercise(P < 0.05). Plasma creatine kinaseconcentrations were 125 ± 25 IU/l (Trn) and 91 ± 9 IU/l (UT)preexercise and 112 ± 14 IU/l (Trn) and 144 ± 22 IU/l(UT; P < 0.05 vs.preexercise) at 48-72 h postexercise (normal: 30-200 IU/l).We conclude that endurance exercise training results in an increasedability to accumulate muscle glycogen after exercise.

     

Extracellular signal-regulated kinase pathway is differentially involved in beta-agonist-induced hypertrophy in slow and fast muscles

The molecular mechanisms controlling -adrenergic receptor agonist (BA)-induced skeletal muscle hypertrophy are not well known. We presently report that BA exerts a distinct muscle- and muscle fiber type-specific hypertrophy. Moreover, we have shown that pharmacologically or genetically attenuating extracellular signal-regulated kinase (ERK) signaling in muscle fibers resulted in decreases (P < 0.05) in fast but not slow fiber type-specific reporter gene expressions in response to BA exposure in vitro and in vivo. Consistent with these data, forced expression of MAPK phosphatase 1, a nuclear protein that dephosphorylates ERK1/2, in fast-twitch skeletal muscle ablated (P < 0.05) the hypertrophic effects of BA feeding (clenbuterol, 20 parts per million in water) in vivo. Further analysis has shown that BA-induced phosphorylation and activation of ERK occurred to a greater (P < 0.05) extent in fast myofibers than in slow myofibers. Analysis of the basal level of ERK activity in slow and fast muscles revealed that ERK1/2 is activated to a greater extent in fast- than in slow-twitch muscles. These data indicate that ERK signaling is differentially involved in BA-induced hypertrophy in slow and fast skeletal muscles, suggesting that the increased abundance of phospho-ERK1/2 and ERK activity found in fast-twitch myofibers, compared with their slow-twitch counterparts, may account, at least in part, for the fiber type-specific hypertrophy induced by BA stimulation. These data suggest that fast myofibers are pivotal in the adaptation of muscle to environmental cues and that the mechanism underlying this change is partially mediated by the MAPK signaling cascade. muscle fiber type; mitogen-activated protein kinase signaling pathways; mechanism     

Regulation of intracellular Ca2+ release in corpus cavernosum smooth muscle: synergism between nitric oxide and cGMP

Tonic contraction of corpus cavernosum smooth muscle cells (SMCs) maintains the flaccid state of the penis, and relaxation is initiated by nitric oxide (NO), leading to erection. Our aim was to investigate the effect of NO on the smooth muscle cellular response to adrenergic stimulation in corpus cavernosum. Fura-2 fluorescence was used to record intracellular Ca²⁺ concentration ([Ca²⁺]_i) from freshly isolated SMCs from rat and human. Phenylephrine (PE) transiently elevated [Ca²⁺]_i in the presence and absence of extracellular Ca²⁺, indicating release from intracellular stores. Whereas the NO donor S-nitroso-N-acetylpenicillamine (SNAP) with sildenafil citrate (SIL) caused no change in basal [Ca²⁺]_i, the PE-induced rise of [Ca²⁺]_i was reversibly inhibited by 27 ± 7% (n = 21, P < 0.005) in rat and by 55 ± 15% (n = 9, P < 0.01) in human SMCs. SNAP and SIL also reduced the contractile response to PE. To investigate the mechanism, we applied mediators alone or in combination. The soluble guanylyl cyclase inhibitor ODQ reduced the effect of SNAP and SIL. SIL, cGMP analogs, and NO donors without SIL did not reduce the PE-induced rise of [Ca²⁺]_i. However, the combination of 8-bromo-cGMP with SNAP reduced the Ca²⁺ peak by 42 ± 9% (n = 22, P < 0.01). Our results demonstrate that NO and cGMP act synergistically to reduce Ca²⁺ release from intracellular stores. Reduction of intracellular Ca²⁺ release may contribute to relaxation of the corpus cavernosum, leading to erection. calcium stores; nitric oxide; sildenafil citrate; inositol 1,4,5-trisphosphate receptor     

Postnatal expression and activity of the mitochondrial 2-oxoglutarate-malate carrier in intact hearts

This studyexamines the functional implications of postnatal changes in theexpression of the mitochondrial transporter protein, 2-oxoglutarate-malate carrier (OMC). Online ¹³C nuclearmagnetic resonance (¹³C NMR) measurements of isotopekinetics in hearts from neonate (3-4 days) and adult rabbitsprovided tricarboxylic acid cycle flux rates and flux rates throughOMC. Neonate and adult hearts oxidizing 2.5 mM[2,4-¹³C₂]butyrate were subjected toeither normal or high cytosolic redox state (2.5 mM lactate) conditionsto evaluate the recruitment of malate-aspartate activity and theresulting OMC flux. During development from neonate (3-4 days) toadult, mitochondrial protein density in the heart increased from19 ± 3% to 31 ± 2%, whereas OMC expression decreased by65% per mitochondrial protein content (P < 0.05).Correspondingly, OMC flux was lower in adults hearts than in neonatesby 73% (neonate = 7.4 ± 0.4, adult = 2.0 ± 0.1 µmol/min per 100 mg mitochondrial protein; P < 0.05). Despite clear changes in OMC content and flux, theresponsiveness of the malate-aspartate shuttle to increased cytosolicNADH was similar in both adults and neonates with an approximatethreefold increase in OMC flux (in densitometric units/100 mgmitochondrial protein: neonate = 25.8 ± 2.5, adult = 6.0 ± 0.2; P < 0.05). The¹³C NMR data demonstrate that OMC activity is a principalcomponent of the rate of labeling of glutamate.