Rat brain VEGF expression in alveolar hypoxia: possible role in high-altitude cerebral edema

The mechanism by which hypoxia causes high-altitudecerebral edema (HACE) is unknown. Tissue hypoxia triggers angiogenesis, initially by expressing vascular endothelial growth factor (VEGF), which has been shown to increase extracerebral capillary permeability. This study investigated brain VEGF expression in 32 rats exposed toprogressively severe normobaric hypoxia (9-6%O₂) for 0 (control), 3, 6, or 12 h or 1, 2, 3, or 6 days. O₂concentration was adjusted intermittently to the limit of tolerance byactivity and intake, but no attempt was made to detect HACE. Northernblot analysis demonstrated that two molecular bands of transcribed VEGFmRNA (~3.9 and 4.7 kb) were upregulated in cortex and cerebellumafter as little as 3 h of hypoxia, with a threefold increase peaking at12-24 h. Western blot revealed that VEGF protein was increased after 12 h of hypoxia, reaching a maximum in ~2 days. The expression of flt-1 mRNA was enhanced after 3 days of hypoxia. We conclude that VEGF production in hypoxia isconsistent with the hypothesis that angiogenesis may be involved inHACE.

     

Differing temporal roles of Ca2+ and cAMP in nicotine-elicited elevation of tyrosine hydroxylase mRNA

The involvement of cAMP- andCa²⁺-mediated pathways in theactivation of tyrosine hydroxylase (TH) gene expression by nicotine wasexamined in PC-12 cells. ExtracellularCa²⁺ and elevations inintracellular Ca²⁺ concentration([Ca²⁺]_i)were required for nicotine to increase TH mRNA. The nicotine-elicited rapid rise in[Ca²⁺]_iwas inhibited by blockers of either L-type or N-type, and to a lesserextent P/Q-, but not T-type, voltage-gatedCa²⁺ channels. With continualnicotine treatment,[Ca²⁺]_ireturned to basal levels within 3-4 min. After a lag of~5-10 min, there was a smaller elevation in[Ca²⁺]_ithat persisted for 6 h and displayed different responsiveness toCa²⁺ channel blockers. This secondphase of elevated[Ca²⁺]_iwas blocked by an inhibitor of store-operatedCa²⁺ channels, consistent with theobserved generation of inositol trisphosphate.1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), when added before or 2 h after nicotine,prevented elevation of TH mRNA. Nicotine treatment significantly raised cAMP levels. Addition of the adenylyl cyclase inhibitor2',5'-dideoxyadenosine (DDA) prevented thenicotine-elicited phosphorylation of cAMP response element bindingprotein. DDA also blocked the elevation of TH mRNA only when addedafter the initial transient rise in [Ca²⁺]_iand not after 1 h. This study reveals that several temporal phases areinvolved in the induction of TH gene expression by nicotine, each ofthem with differing requirements forCa²⁺ and cAMP.

     

Hypoxia induces permeability in brain microvessel endothelial cells via VEGF and NO

In this study, an in vitro model of the blood-brain barrier,consisting of porcine brain-derived microvascular endothelial cells(BMEC), was used to evaluate the mechanism of hypoxia-induced hyperpermeability. We show that hypoxia-induced permeability in BMECwas completely abolished by a neutralizing antibody to vascular endothelial growth factor (VEGF). In contrast, under normoxic conditions, addition of VEGF up to 100 ng/ml did not alter monolayer barrier function. Treatment with either hypoxia or VEGF under normoxicconditions induced a twofold increase in VEGF binding sites and VEGFreceptor 1 (Flt-1) mRNA expression in BMEC. Hypoxia-induced permeability also was prevented by the nitric oxide (NO) synthase inhibitor N^G-monomethyl-L-arginine,suggesting that NO is involved in hypoxia-induced permeability changes,which was confirmed by measurements of the cGMP level. During normoxia,treatment with VEGF (5 ng/ml) increased permeability as well as cGMPcontent in the presence of several antioxidants. These results suggestthat hypoxia-induced permeability in vitro is mediated by the VEGF/VEGFreceptor system in an autocrine manner and is essentially dependent onreducing conditions stabilizing the second messenger NO as the mediatorof changes in barrier function of BMEC.     

Activity and protein localization of multiple glutamate transporters in gestation day 14vs. day 20rat placenta

Concentrative absorption of glutamate by the developing placentais critical for proper fetal development. The expression of GLAST1,GLT1, EAAC1, and EAAT4, known to be capable ofD-aspartate-inhibitable andNa⁺-coupled glutamate transport(system ), was evaluated inday 14 vs. day20 rat chorioallantoic placenta. Steady-state mRNAlevels were greater at day 20 for alltransporters. Immunohistochemistry determined that the expression ofGLAST1, GLT1, and EAAC1 was greater throughout the day20 placenta and was asymmetric with respect to cellularlocalization. EAAT4 protein was not detected. System activity was responsible formost of the Na⁺-dependentglutamate uptake and was greater in day20 than in day 14 apical and basal membrane subdomains of the labyrinthsyncytiotrophoblast. Greater quantities of EAAC1 and GLAST1 proteinwere identified on day 20, andquantities were greater in basal than in apical membranes. GLT1expression, unchanged in apical membranes, was decreased in basalmembranes. These data correlate transporter mRNA and protein contentwith transport activity and demonstrate an increasing capacity forglutamate absorption by the developing placenta.

     

Age-dependent response of the electrocardiogram to K(+) channel blockers in mice

Developmental changes in electrocardiogram (ECG) andresponse to selective K⁺ channelblockers were assessed in conscious, unsedated neonatal (days 1, 7, 14) and adult male mice(>60 days of age). Mean sinus R-R interval decreased from 120 ± 3 ms in day 1 to 110 ± 3 ms inday 7, 97 ± 3 ms inday 14, and 81 ± 1 ms in adultmice (P < 0.001 by ANOVA; all 3 groups different from day 1). Inparallel, the mean P-R interval progressively decreased duringdevelopment. Similarly, the mean Q-T interval decreased from 62 ± 2 ms in day 1 to 50 ± 2 ms inday 7, 47 ± 8 ms inday 14 neonatal mice, and 46 ± 2 ms in adult mice (P < 0.001 byANOVA; all 3 groups are significantly different fromday 1).Q-T_c was calculated asQ- interval.Q-T_c significantly shortened from179 ± 4 ms in day 1 to 149 ± 5 ms in day 7 mice(P < 0.001). In addition, the J junction-S-T segment elevation observed in day1 neonatal mice resolved by day14. Dofetilide (0.5 mg/kg), the selective blocker ofthe rapid component of the delayed rectifier(I_Kr) abolished S-T segment elevation and prolonged Q-T andQ-T_c intervals in day 1 neonates but not in adult mice.In contrast, 4-aminopyridine (4-AP, 2.5 mg/kg) had no effect onday 1 neonates but in adults prolongedQ-T and Q-T_c intervals andspecifically decreased the amplitude of a transiently repolarizingwave, which appears as an r' wave at the end of the apparent QRSin adult mice. In conclusion, ECG intervals and configuration changeduring normal postnatal development in the mouse.K⁺ channel blockers affect themouse ECG differently depending on age. These data are consistent withthe previous findings that the dofetilide-sensitiveI_Kr is dominantin day 1 mice, whereas 4-AP-sensitivecurrents, the transiently repolarizingK⁺ current, and the rapidlyactivating, slowly inactivating K⁺current are the dominant K⁺currents in adult mice. This study provides background information useful for assessing abnormal development in transgenic mice.

     

Exercise training increases sarcolemmal GLUT-4 protein and mRNA content in diabetic heart

Osborn, Brett A., June T. Daar, Richard A. Laddaga, Fred D. Romano, and Dennis J. Paulson. Exercise training increases sarcolemmal GLUT-4 protein and mRNA content in diabetic heart. J. Appl. Physiol. 82(3): 828-834, 1997.This study determined whether dynamic exercise training ofdiabetic rats would increase the expression of the GLUT-4 glucosetransport protein in prepared cardiac sarcolemmal membranes. Fourgroups were compared: sedentary control, sedentary diabetic, trainedcontrol, and trained diabetic. Diabetes was induced by intravenousstreptozotocin (60 mg/kg). Trained control and diabetic rats were runon a treadmill for 60 min, 27 m/min, 10% grade, 6 days/wk for 10 wk.Sarcolemmal membranes were isolated by using differentialcentrifugation, and the activity of sarcolemmalK⁺-p-nitrophenylphosphatase( pNPPase; an indicator ofNa⁺-K⁺-adenosinetriphosphataseactivity) was quantified. Hearts from the sedentary diabetic groupexhibited a significant depression of sarcolemmal pNPPaseactivity. Exercise training did not significantly alterpNPPase activity. Sedentary diabetic rats exhibited an 84 and 58% decrease in GLUT-4 protein and mRNA, respectively, relative tocontrol rats. In the trained diabetic animals, sarcolemmal GLUT-4protein levels were only reduced by 50% relative to control values,whereas GLUT-4 mRNA were returned to control levels. The increase inmyocardial sarcolemmal GLUT-4 may be beneficial to the diabetic heartby enhancing myocardial glucose oxidation and cardiac performance

     

Caco-2 intestinal cell differentiation is associated with G1 arrest and suppression of CDK2 and CDK4

The cellular mechanisms regulating intestinal proliferation anddifferentiation remain largely undefined. Previously, we showed anearly induction of the cyclin-dependent kinase (CDK) inhibitor p21^Waf1/Cip1 in Caco-2 cells, ahuman colon cancer line that spontaneously differentiates into a smallbowel phenotype. The purpose of our present study was to assess thetiming of cell cycle arrest in relation to differentiation in Caco-2cells and to examine the mechanisms responsible for CDK inactivation.Caco-2 cells undergo a relativeG₁/S block and cease toproliferate at day3 postconfluency; an increase in theactivity of terminally differentiated brush-border enzymes (sucrase andalkaline phosphatase) was noted at day6 postconfluency. Cell cycle block wasassociated with suppression of both CDK2 and CDK4 activities, which areimportant for G₁/S progression.Treatment of the CDK immune complexes with the detergent deoxycholate(DOC) resulted in restoration of CDK2, but not CDK4, activity atday 3 postconfluency, suggesting the presence of inhibitory protein(s)binding to the cyclin/CDK2 complex at this time point. An increasedbinding of p21^Waf1/Cip1 to CDK2complexes at day3 postconfluency was noted, suggesting a potential role for p21^Waf1/Cip1in CDK2 inactivation; however, immunodepletion ofp21^Waf1/Cip1 from Caco-2 proteinextracts demonstrated thatp21^Waf1/Cip1 is only partiallyresponsible for CDK2 suppression atday 3 postconfluency. A decrease in the cyclin E/CDK2 complex appears tocontribute to the CDK2 inactivation noted atdays6 and12 postconfluency. Taken together, ourresults suggest that multiple mechanisms contribute to CDK suppressionduring Caco-2 cell differentiation. Inhibition of CDK2 and CDK4 leadsto G₁ arrest and inhibition ofproliferation that precede Caco-2 cell differentiation.

     

Myogenic regulatory factors during regeneration of skeletal muscle in young, adult, and old rats

Marsh, Daniel R., David S. Criswell, James A. Carson, andFrank W. Booth. Myogenic regulatory factors during regeneration ofskeletal muscle in young, adult, and old rats. J. Appl. Physiol. 83(4): 1270-1275, 1997.Myogenicfactor mRNA expression was examined during muscle regeneration afterbupivacaine injection in Fischer 344/Brown Norway F1 rats aged 3, 18, and 31 mo of age (young, adult, and old, respectively). Mass of thetibialis anterior muscle in the young rats had recovered to controlvalues by 21 days postbupivacaine injection but in adult and old ratsremained 40% less than that of contralateral controls at 21 and 28 days of recovery. During muscle regeneration, myogenin mRNA wassignificantly increased in muscles of young, adult, and old rats 5 daysafter bupivacaine injection. Subsequently, myogenin mRNA levels inyoung rat muscle decreased to postinjection control values byday 21 but did not return to controlvalues in 28-day regenerating muscles of adult and old rats. Theexpression of MyoD mRNA was also increased in muscles atday 5 of regeneration in young, adult,and old rats, decreased to control levels by day14 in young and adult rats, and remained elevated inthe old rats for 28 days. In summary, either a diminished ability todownregulate myogenin and MyoD mRNAs in regenerating muscle occurs inold rat muscles, or the continuing myogenic effort includes elevatedexpression of these mRNAs.

     

Depressed ventilatory response to hypoxia in hypothermic newborn piglets: role of glutamate

To evaluatewhether changes in extracellular glutamate (Glu) levels in the centralnervous system could explain the depressed hypoxic ventilatory responsein hypothermic neonates, 12 anesthetized, paralyzed, and mechanicallyventilated piglets <7 days old were studied. The Glu levels in thenucleus tractus solitarius obtained by microdialysis, minute phrenicoutput (MPO), O₂ consumption, arterial blood pressure, heart rate, and arterial blood gases weremeasured in room air and during 15 min of isocapnic hypoxia (inspiredO₂ fraction = 0.10) at braintemperatures of 39.0 ± 0.5°C [normothermia (NT)]and 35.0 ± 0.5°C [hypothermia (HT)]. During NT, MPO increased significantly during hypoxia and remained above baseline. However, during HT, there was a marked decrease in MPOduring hypoxia (NT vs. HT, P < 0.03). Glu levels increased significantly in hypoxia during NT;however, this increase was eliminated during HT(P < 0.02). A significant linearcorrelation was observed between the changes in MPO and Glu levelsduring hypoxia (r = 0.61, P < 0.0001). Changes in pH, arterialPO₂, O₂ consumption, arterial bloodpressure, and heart rate during hypoxia were not different between theNT and HT groups. These results suggest that the depressed ventilatoryresponse to hypoxia observed during HT is centrally mediated and inpart related to a decrease in Glu concentration in the nucleus tractussolitarius.

     

Physiological and Genomic Consequences of Intermittent Hypoxia: Selected Contribution: Altered vascular reactivity in arterioles of chronic intermittent hypoxic rats

Recurrentepisodic hypoxia (EH) is a feature of sleep apnea that may beresponsible for some chronic cardiovascular sequelae such as systemichypertension. Chronic EH (8 h/day for 35 days) causes elevation ofdiurnal resting (unstimulated) mean arterial blood pressure (MAP) inthe rat. We used in vivo video microscopy to examine arteriolarreactivity in the cremaster muscle of male Sprague-Dawley ratssubjected to 35 days of EH. Cremaster muscles of EH (n = 6) and control (n = 6) rats were exposed to varying doses of norepinephrine (NE) (10¹⁰ to 10⁵M), ACh (10⁹ to 10⁵ M), and endothelin-1(10¹² to 10⁸ M). In a separate experiment,EH (n = 5) and control (n = 6) ratswere given one dose of a nitric oxide synthase (NOS) inhibitor N^G-nitro-L-arginine methylester (L-NAME; 10⁵ M). We also examinedendothelial NOS mRNA from the kidneys of EH-stimulated and control(unstimulated) rats. Telemetry-monitored EH rats showed a 16-mmHgincrease in MAP over 35 days, whereas control rats showed no change.The response to NE and endothelin-1 were similar for EH and controlrats. ACh vasodilatation of arterioles in EH rats was significantlyattenuated compared with that of controls. The degree ofvasoconstriction in response to blockade of the nitric oxide system byL-NAME was significantly less (83% of baseline diameterwith L-NAME) for arterioles of EH rats compared with thatfor controls (61% of baseline diameter), implying lower basal restingnitric oxide release in the EH rats. Whole kidney mRNA endothelial NOSlevels were not different between groups. These data support thehypothesis that chronic elevation of blood pressure associated with EHinvolves increased peripheral resistance from decreased basal releaseor production of nitric oxide after 35 days of EH.

     

Physiological and Genomic Consequences of Intermittent Hypoxia: Selected Contribution: Chemoreflex responses to CO2 before and after an 8-h exposure to hypoxia in humans

The ventilatorysensitivity to CO₂, in hyperoxia, is increased after an 8-hexposure to hypoxia. The purpose of the present study was to determinewhether this increase arises through an increase in peripheral orcentral chemosensitivity. Ten healthy volunteers each underwent 8-hexposures to 1) isocapnic hypoxia, with end-tidalPO₂ (PET_O2) = 55 Torr and end-tidal PCO₂(PET_CO2) = eucapnia; 2)poikilocapnic hypoxia, with PET_O2 = 55 Torr and PET_CO2 = uncontrolled;and 3) air-breathing control. The ventilatory response toCO₂ was measured before and after each exposure with theuse of a multifrequency binary sequence with two levels of PET_CO2: 1.5 and 10 Torr above the normalresting value. PET_O2 was held at 250 Torr.The peripheral (Gp) and the central (Gc) sensitivities were calculatedby fitting the ventilatory data to a two-compartment model. There wereincreases in combined Gp + Gc (26%, P < 0.05),Gp (33%, P < 0.01), and Gc (23%, P = not significant) after exposure to hypoxia. There were no significant differences between isocapnic and poikilocapnic hypoxia. We conclude that sustained hypoxia induces a significant increase inchemosensitivity to CO₂ within the peripheral chemoreflex.

     

Perinatal nicotine exposure impairs ability of newborn rats to autoresuscitate from apnea during hypoxia

Failure toautoresuscitate by hypoxic gasping during prolonged sleep apnea hasbeen suggested to play a role in sudden infant death. Furthermore,maternal smoking has been repeatedly shown to be a risk factor forsudden infant death. The present experiments were carried out onnewborn rat pups to investigate the influence of perinatal exposure tonicotine (the primary pharmacological and addictive agent in tobacco)on their time to last gasp during a single hypoxic exposure and ontheir ability to autoresuscitate during repeated exposure to hypoxia.Pregnant rats received either nicotine (6 mg · kg¹ · 24 h¹) or vehiclecontinuously from day 6 of gestationto days 5 or 6 postpartum via an osmotic minipump.On days 5 or6 postpartum, pups were exposed eitherto a single period of hypoxia (97%N₂-3% CO₂) and their time to last gaspwas determined, or they were exposed repeatedly to hypoxia and theirability to autoresuscitate from primary apnea was determined. Perinatalexposure to nicotine did not alter the time to last gasp, but it didimpair the ability of pups to autoresuscitate from primary apnea. Aftervehicle, the pups were able to autoresuscitate from 18 ± 1 (SD)periods of hypoxia, whereas, after nicotine, the pups were able toautoresuscitate from only 12 ± 2 periods(P < 0.001) of hypoxia. Thus ourdata provide evidence that perinatal exposure to nicotine impairs the ability of newborn rats to autoresuscitate from primary apnea duringrepeated exposure to hypoxia, such as may occur during episodes ofprolonged sleep apnea.

     

Serum response factor mRNA induction in the hypertrophying chicken patagialis muscle

Gene expression inthe stretched chicken patagialis (Pat) muscle has not been extensivelyexamined. This study's purpose was to determine the Pat muscle'sexpression pattern of serum response factor (SRF), skeletal -actin,and MyoD mRNAs after 3 days (onset of stretch), 6 days (end of firstweek of rapid growth), and 14 days (slowed rate of stretch-inducedgrowth) of stretch. SRF mRNA demonstrated two species (B1 and B2), withB2 being more prevalent in the predominantly fast-twitch Pat muscle,compared with the slow-tonic muscle. Stretch overload increased B1 andB2 SRF mRNA concentrations, and the increase in B1 SRF mRNAconcentration was greater at day 6 compared with days 3 or14. MyoD mRNA concentration wasgreater in 3-day-stretched Pat muscles, compared withdays 6 or14 . Skeletal -actin mRNAconcentration was not changed during the study. Gel mobility shiftassays demonstrated that SRF binding with serum response element 1 ofthe skeletal -actin promoter had no altered binding patterns from6-day-stretched Pat nuclear extracts. It appears that SRF and MyoDmRNAs are induced in the stretch-overloaded Pat muscle but at differenttime points.

     

Enhanced expression of the Flt‐1 and Flk‐1 receptor tyrosine kinases in a newborn piglet model of ischemic tolerance

Vascular endothelial growth factor (VEGF), an angiogenic factor induced by hypoxia, also exerts direct effects on neural tissues. VEGF up‐regulation after hypoxia coincides with expression of its two tyrosine kinase receptors Flt‐1(VEGFR‐1) and Flk‐1 (KDR/VEGFR‐2), which are the key mediators of physiological angiogenesis. We have recently shown that hypoxic‐preconditioning (PC) leading to tolerance to hypoxia–ischemia in neonatal piglet brain resulted in increased expression of VEGF. In this study, we used a hypoxic‐preconditioning model of ischemic tolerance to analyze the expression and cellular distribution of VEGF receptors and phosphorylation of cAMP‐response element‐binding protein (CREB) in newborn piglet brain. The response of Flt‐1 and Flk‐1 mRNA to PC alone was biphasic with peaks early (6 h) and late (1 week) after PC. The mRNA expression of Flt‐1 and Flk‐1 in piglets preconditioned 24 h prior to hypoxia–ischemia was significantly higher than non‐preconditioned piglets and remained up‐regulated up to 7 days. Furthermore, PC prior to hypoxia–ischemia significantly increased the protein levels of Flt‐1 and Flk‐1 compared with hypoxia–ischemia in a time‐dependent manner. Double‐immunolabeling indicated that both Flt‐1 and Flk‐1 are expressed in neurons and endothelial cells with a similar time course of expression following PC and that PC leads to the growth of new vessels. Finally, our data demonstrate that PC significantly phosphorylated and activated cAMP‐response element‐binding protein in nucleus. These results suggest that mechanism(s) initiated by PC can induce VEGF receptor up‐regulation in newborn brain and that VEGF–VEGF receptor‐coupled signal transduction pathways could contribute to the establishment of tolerance following hypoxia–ischemia.     

Regional clearance of solute from peripheral airway epithelia: recovery after sublobar exposure to ozone

The influence oflocal exposure to ozone (O₃) onrespiratory epithelial permeability of sublobar lung segments wasstudied by using aerosolized^99mTc-diethylenetriaminepentaacetic acid (DTPA; mol wt, 492). Two bronchoscopes were insertedthrough an endotracheal tube in anesthetized, mechanically ventilated,mixed breed dogs and were wedged into sublobar bronchi located in theright and left lower lobes, respectively. Segments were ventilated viathe bronchoscope with 5% CO₂ inair delivered at 200 ml/min, and an aerosol of^99mTc-DTPA was generated anddelivered through the scope and into the sublobar segment over a 30-speriod. Clearance of ^99mTc-DTPAwas measured simultaneously from right and left lower lung segments atbaseline and 1, 7, and 14 days after a 6-h sublobar exposure tofiltered air or 400 parts per billionO₃.O₃ treatment significantlydecreased the clearance halftime(t₅₀) of^99mTc-DTPA by 50% from thebaseline mean of 32.3 to 16.0 min at 1 day postexposure. After 7 daysof recovery, t₅₀was still reduced by 28.8%; however, by 14 days postexposure,clearance of ^99mTc-DTPA hadrecovered, and thet₅₀ had a meanvalue of 30.0 min. ^99mTc-DTPAclearance was not altered by exposure to filtered air, andt₅₀ values werecomparable to baseline at 1, 7, and 14 days postexposure. These resultsreveal that a single local exposure toO₃ increases transepithelialclearance, but only for epithelia directly exposed toO₃, and that 7-14 days ofrecovery are required before permeability to small-molecular-weightsolutes returns to normal.     

Age-related differences in Na+-dependent Ca2+ accumulation in rabbit hearts exposed to hypoxia and acidification

In this study, we test the hypothesisthat in newborn hearts (as in adults) hypoxia and acidificationstimulate increased Na⁺ uptake, in part via pH-regulatoryNa⁺/H⁺ exchange. Resulting increases inintracellular Na⁺ (Na_i) alter the force drivingthe Na⁺/Ca²⁺ exchanger and lead to increasedintracellular Ca²⁺. NMR spectroscopy measuredNa_i and cytosolic Ca²⁺ concentration([Ca²⁺]_i) and pH (pH_i) inisolated, Langendorff-perfused 4- to 7-day-old rabbit hearts. AfterNa⁺/K⁺ ATPase inhibition, hypoxic hearts gainedNa⁺, whereas normoxic controls did not [19 ± 3.4 to139 ± 14.6 vs. 22 ± 1.9 to 22 ± 2.5 (SE) meq/kg drywt, respectively]. In normoxic hearts acidified using theNH₄Cl prepulse, pH_i fell rapidly and recovered,whereas Na_i rose from 31 ± 18.2 to 117.7 ± 20.5 meq/kg dry wt. Both protocols caused increases in [Ca]_i;however, [Ca]_i increased less in newborn hearts than inadults (P < 0.05). Increases in Na_i and[Ca]_i were inhibited by theNa⁺/H⁺ exchange inhibitormethylisobutylamiloride (MIA, 40 µM; P < 0.05), aswell as by increasing perfusate osmolarity (+30 mosM) immediately before and during hypoxia (P < 0.05). The data supportthe hypothesis that in newborn hearts, like adults, increases inNa_i and [Ca]_i during hypoxia and afternormoxic acidification are in large part the result of increased uptakevia Na⁺/H⁺ and Na⁺/Ca²⁺exchange, respectively. However, for similar hypoxia and acidification protocols, this increase in [Ca]_i is less in newborn thanadult hearts.

     

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.

     

Aldosterone induces ras methylation in A6 epithelia

Aldosterone increases Na⁺ reabsorption by renalepithelial cells: the acute actions (<4 h) appear to be promoted byprotein methylation. This paper describes the relationship betweenprotein methylation and aldosterone's action and describesaldosterone-mediated targets for methylation in cultured renal cells(A6). Aldosterone increases protein methylation from 7.90 ± 0.60 to 20.1 ± 0.80 methyl ester cpm/µg protein. Aldosteronestimulates protein methylation by increasing methyltransferase activityfrom 14.0 ± 0.64 in aldosterone-depleted cells to 31.8 ± 2.60 methyl ester cpm/µg protein per hour in aldosterone-treated cells. Three known methyltransferase inhibitors reduce thealdosterone-induced increase in methyltransferase activity. One ofthese inhibitors, the isoprenyl-cysteine methyltransferase-specificinhibitor,S-trans,trans-farnesylthiosalicylic acid, completely blocks aldosterone-induced protein methylation and also aldosterone-induced short-circuit current. Aldosterone inducesprotein methylation in two molecular weight ranges: near 90 kDa andaround 20 kDa. The lower molecular weight range is the weight of smallG proteins, and aldosterone does increase both Ras protein 1.6-fold andRas methylation almost 12-fold. Also, Ras antisense oligonucleotidesreduce the activity of Na⁺ channels by about fivefold. Weconclude that 1) protein methylation is essential foraldosterone-induced increases in Na⁺ transport;2) one target for methylation is p21^ras; and3) inhibition of Ras expression or Ras methylation inhibits Na⁺ channel activity.

     

Zinc status affects p53, gadd45, and c-fos expression and caspase-3 activity in human bronchial epithelial cells

This study was designed to examine theinfluence of zinc depletion and supplementation on the expression ofp53 gene, target genes of p53, andcaspase-3 activity in normal human bronchial epithelial (NHBE) cells. Aserum-free, low-zinc medium containing 0.4 µmol/l of zinc [zincdeficient (ZD)] was used to deplete cellular zinc over one passage. Inaddition, cells were cultured for one passage in media containing 4.0 µmol/l of zinc [zinc normal (ZN)], which represents normal cultureconcentrations (Clonetics); 16 µmol/l of zinc [zinc adequate (ZA)],which represents normal human plasma zinc levels; or 32 µmol/l ofzinc [zinc supplemented (ZS)], which represents the high end ofplasma zinc levels attainable by oral supplementation in humans.Compared with ZN cells, cellular zinc levels were 76% lower in ZDcells but 3.5-fold and 6-fold higher in ZA and ZS cells, respectively.Abundances of p53 mRNA and nuclear p53 protein were elevatedin treatment groups compared with controls (ZN). For p53mRNA abundance, the highest increase (3-fold) was observed in ZD cells.In contrast, the highest increase (17-fold) in p53 nuclearprotein levels was detected in ZS cells. Moreover, gadd45mRNA abundance was moderately elevated in ZD and ZA cells and was notaltered in ZS cells compared with ZN cells. Furthermore, the onlyalteration in c-fos mRNA and caspase-3 activity was thetwofold increase and the 25% reduction, respectively, detected in ZScompared with ZN cells. Thus p53, gadd45, andc-fos and caspase-3 activity appeared to be modulated bycellular zinc status in NHBE cells.