Nitric oxide-induced inhibition of smooth muscle cell proliferation involves S-nitrosation and inactivation of RhoA

Nitric oxide (NO) acts as a vasoregulatory molecule that inhibits vascular smooth muscle cell (SMC) proliferation. Studies have illustrated that NO inhibits SMC proliferation via the extracellular signal-regulated kinase (ERK) pathway, leading to increased protein levels of the cyclin-dependent kinase inhibitor p21^Waf1/Cip1. The ERK pathway can be pro- or antiproliferative, and it has been demonstrated that the activation status of the small GTPase RhoA determines the proliferative fate of ERK signaling, whereby inactivation of RhoA influences ERK signaling to increase p21^Waf1/Cip1 and inhibit proliferation. The purpose of these investigations was to examine the effect of NO on RhoA activation/S-nitrosation and to test the hypothesis that inhibition of SMC proliferation by NO is dependent on inactivation of RhoA. NO decreases activation of RhoA, as demonstrated by RhoA GTP-binding assays, affinity precipitation, and phalloidin staining of the actin cytoskeleton. Additionally, these effects are independent of cGMP. NO decreases SMC proliferation, and gene transfer of constitutively active RhoA (RhoA^63L) diminished the antiproliferative effects of NO, as determined by thymidine incorporation. Western blots of p21^Waf1/Cip1 correlated with changes in proliferation. S-nitrosation of recombinant RhoA protein and immunoprecipitated RhoA was demonstrated by Western blotting for nitrosocysteine and by measurement of NO release. Furthermore, NO decreases GTP loading of recombinant RhoA protein. These findings indicate that inactivation of RhoA plays a role in NO-mediated SMC antiproliferation and that S-nitrosation is associated with decreased GTP binding of RhoA. Nitrosation of RhoA and other proteins likely contributes to cGMP-independent effects of NO. cell signaling; posttranslational modification; vascular disease     

Mechanical and metabolic determination of VO2 and fatigue during repetitive isometric contractions in situ

Repetitiveisometric tetanic contractions (1/s) of the caninegastrocnemius-plantaris muscle were studied either at optimal length(L_o) or shortlength (L_s;~0.9 · L_o),to determine the effects of initial length on mechanical and metabolicperformance in situ. Respective averages of mechanical and metabolicvariables were(L_o vs.L_s, allP < 0.05) passive tension (preload) = 55 vs. 6 g/g, maximal active tetanic tension(P_o) = 544 vs. 174 (0.38 · P_o)g/g, maximal blood flow () = 2.0 vs. 1.4 ml · min¹ · g¹,and maximal oxygen uptake(O₂) = 12 vs. 9 µmol · min¹ · g¹.Tension at L_odecreased to0.64 · P_o over20 min of repetitive contractions, demonstrating fatigue; there were nosignificant changes in tension atL_s. In separatemuscles contracting atL_o, was set to that measured atL_s (1.1 ml · min¹ · g¹),resulting in decreased O₂(7 µmol · min¹ · g¹),and rapid fatigue, to0.44 · P_o. Thesedata demonstrate that 1)muscles at L_ohave higher andO₂ values than those at L_s;2) fatigue occurs atL_o with highO₂, adjusting metabolic demand (tension output) to match supply; and3) the lack of fatigue atL_s with lowertension, , andO₂ suggestsadequate matching of metabolic demand, set low by shortmuscle length, with supply optimized by low preload. Thesedifferences in tension andO₂ betweenL_o andL_s groupsindicate that muscles contracting isometrically at initial lengthsshorter than L_oare working under submaximal conditions.

     

Endogenous expression of the renal high-affinity H+-peptide cotransporter in LLC-PK1 cells

The reabsorption of filtered di- andtripeptides as well as certain peptide mimetics from the tubular lumeninto renal epithelial cells is mediated by anH⁺-coupledhigh-affinity transport process. Here we demonstrate for the first timeH⁺-coupled uptake of dipeptidesinto the renal proximal tubule cell lineLLC-PK₁. Transport was assessed1) by uptake studies using theradiolabeled dipeptideD-[³H]Phe-L-Ala,2) by cellular accumulation of the fluorescent dipeptide D-Ala-Lys-AMCA, and3) by measurement of intracellularpH (pH_i) changes as aconsequence of H⁺-coupleddipeptide transport. Uptake ofD-Phe-L-Alaincreased linearly over 11 days postconfluency and showed all thecharacteristics of the kidney cortex high-affinity peptide transporter,e.g., a pH optimum for transport ofD-Phe-L-Alaof 6.0, an apparent K_m value forinflux of 25.8 ± 3.6 µM, and affinities of differently chargeddipeptides or the -lactam antibiotic cefadroxil to the binding sitein the range of 20-80 µM.pH_i measurements established thepeptide transporter to induce pronounced intracellular acidification inLLC-PK₁ cells and confirm itspostulated role as a cellular acid loader.

     

Ca2+ influx inhibits voltage-dependent and augments Ca2+-dependent K+ currents in arterial myocytes

These experiments were performed to determine the effects ofreducing Ca²⁺ influx(Ca_in) onK⁺ currents(I_K) inmyocytes from rat small mesenteric arteries by1) adding externalCd²⁺ or2) lowering externalCa²⁺ to 0.2 mM. When measured froma holding potential (HP) of 20 mV(I_K20),decreasing Ca_in decreasedI_K at voltageswhere it was active (>0 mV). When measured from a HP of 60 mV(I_K60),decreasing Ca_in increasedI_K at voltagesbetween 30 and +20 mV but decreased I_K at voltagesabove +40 mV. Difference currents(I_K) weredetermined by digital subtraction of currents recorded under controlconditions from those obtained whenCa_in was decreased. At testvoltages up to 0 mV,I_K60 exhibitedkinetics similar to controlI_K60, with rapidactivation to a peak followed by slow inactivation. At 0 mV, peakI_K60 averaged75 ± 13 pA (n = 8) withCd²⁺ and 120 ± 20 pA(n = 9) with lowCa²⁺ concentration. At testvoltages from 0 to +60 mV,I_K60 always had an early positive peak phase, but its apparent "inactivation" increased with voltage and its steady value became negative above +20mV. At +60 mV, the initial peakI_K60 averaged115 ± 18 pA with Cd²⁺ and 187 ± 34 pA with low Ca²⁺. With 10 mM pipette BAPTA, Cd²⁺ produced asmall inhibition ofI_K20 but stillincreased I_K60 between 30 and +10 mV. InCa²⁺-free external solution,Cd²⁺ only decreased bothI_K20 andI_K60. In thepresence of iberiotoxin (100 nM) to inhibitCa²⁺-activatedK⁺ channels(K_Ca),Cd²⁺ increasedI_K60 at allvoltages positive to 30 mV while BAY K 8644 (1 µM) decreasedI_K60. Theseresults suggest that Ca_in, through L-type Ca²⁺ channels and perhapsother pathways, increases K_Ca(i.e., I_K20) and decreases voltage-dependent K⁺currents in this tissue. This effect could contribute to membrane depolarization and force maintenance.

     

Uptake and intracellular fate of L-DOPA in a human intestinal epithelial cell line: Caco-2

The aim of the presentstudy was to examine the kinetic characteristics of theL-3,4-dihydroxyphenylalanine (L-DOPA)transporter and the fate of newly formed dopamine in Caco-2 cells. Inthe presence of 50 µM benserazide (an inhibitor of aromaticL-amino acid decarboxylase), L-DOPA was rapidlyaccumulated in Caco-2 cells. At equilibrium (30 min of incubation) theintracellular L-DOPA concentration was 10.2 ± 0.1 µM ata medium concentration of 0.5 µM. In saturation experiments theaccumulation of L-DOPA was saturable with aMichaelis-Menten constant (K_m) of 60 ± 10 µMand a maximal reaction velocity (V_max) of 6.6 ± 0.3 nmol · mg protein¹ · 6 min¹; at 4°C the amount of L-DOPAaccumulated in the cells was nonsaturable. When cells were incubatedwith increasing concentrations of L-DOPA (10-100 µM)in the absence of benserazide, a substantial amount of theL-DOPA that was taken up was decarboxylated to dopamine, with an apparent K_m of 27.2 µM. In experimentsperformed in cells cultured in polycarbonate filters, theaccumulation of L-DOPA in the presence of benserazide wasgreater when the substrate was applied from the basolateral cell borderthan when it was applied from the apical cell border. In the absence ofbenserazide, L-DOPA applied from the basolateral cellborder resulted in a nonlinear formation of dopamine(K_m = 43 ± 7 µM,V_max = 23.7 ± 1.2 nmol · mgprotein¹ · 6 min¹). Theamount of dopamine leaving the cell through the apical cell border waslower than the amount that escaped through the basolateral cell border,and the process was saturable (K_m = 623 ± 238 µM, V_max = 0.19 ± 0.02 nmol · mgprotein¹ · 6 min¹). Inconclusion, the data presented here show that Caco-2 cells are endowedwith an efficient L-DOPA uptake system, and intracellular L-DOPA was found to be rapidly converted to dopamine, someof which diffuses out of the cell. The utilization of Caco-2 cells cultured on polycarbonate filters probably provides a better way tolook at processes such as the outward transfer of intracellular molecules, namely, the outward transfer of newly formed dopamine.

     

Development of ventilatory responsiveness to progressive hypoxia and hypercapnia in low-birth-weight lambs

Moss, T. J., M. G. Davey, G. J. McCrabb, and R. Harding.Development of ventilatory responsiveness to progressive hypoxia and hypercapnia in low-birth-weight lambs. J. Appl.Physiol. 81(4): 1555-1561, 1996.Our aim was todetermine the effects of low birth weight on ventilatory responses toprogressive hypoxia and hypercapnia during early postnatal life. Sevenlow-birth-weight (2.7 ± 0.3 kg) and five normal-birth-weight (4.8 ± 0.2 kg) lambs, all born at term, underwent weekly rebreathingtests during wakefulness while arterialPO₂,PCO₂, and pH were measured. Hypoxicventilatory responsiveness (HOVR; percent increase in ventilation whenarterial PO₂ fell to 60% of resting values) increased in normal lambs from 86.6 ± 7.1% atweek 1 to 227.4 ± 24.9% atweek 6. In low-birth-weight lambs,HOVR was not significantly different at week1 (60.1 ± 18.7%) from that of normal lambs but didnot increase with postnatal age (56.6 ± 19.3% atweek 6). HOVR of all lambs at 6 wkwas significantly correlated with birth weight(r² = 0.8).Hypercapnic ventilatory responsiveness (gradient of ventilation vs.arterial PCO₂) did not change withage and was not significantly different between groups [84.7 ± 7.5 (low-birth-weight lambs) vs. 89.4 ± 6.6 ml ^· min^{1 ·} kg^{1 ·} mmHg¹(normal lambs)]. We conclude that intrauterine conditions that impair fetal growth lead to the failure of HOVR to increase with age.

     

Transport of thiamine in human intestine: mechanism and regulation in intestinal epithelial cell model Caco-2

The present studyexamined the intestinal uptake of thiamine (vitaminB₁) using the human-derivedintestinal epithelial cells Caco-2 as an in vitro model system.Thiamine uptake was found to be 1)temperature and energy dependent and occurred with minimal metabolicalteration; 2) pH sensitive;3)Na⁺ independent;4) saturable as a function ofconcentration with an apparent Michaelis-Menten constant of 3.18 ± 0.56 µM and maximal velocity of 13.37 ± 0.94 pmol · mgprotein¹ · 3 min¹;5) inhibited by the thiaminestructural analogs amprolium and oxythiamine, but not by unrelatedorganic cations tetraethylammonium, N-methylnicotinamide, and choline; and6) inhibited in a competitive mannerby amiloride with an inhibition constant of 0.2 mM. The role ofspecific protein kinase-mediated pathways in the regulation of thiamineuptake by Caco-2 cells was also examined using specific modulators ofthese pathways. The results showed possible involvement of aCa²⁺/calmodulin (CaM)-mediatedpathway in the regulation of thiamine uptake. No role for proteinkinase C- and protein tyrosine kinase-mediated pathways in theregulation of thiamine uptake was evident. These results demonstratethe involvement of a carrier-mediated system for thiamine uptake byCaco-2 intestinal epithelial cells. This system isNa⁺ independent and is differentfrom the transport systems of organic cations. Furthermore, aCaM-mediated pathway appears to play a role in regulating thiamineuptake in these cells.

     

1,25-Dihydroxycholecalciferol enhances butyrate-induced p21(Waf1/Cip1) expression

Butyrate, a short-chain fatty acid produced in the colon, as well as its prodrug tributyrin, reduce proliferation and increase differentiation of colon cancer cells. p21(Waf1/Cip1) and p27(Kip1) are negative regulators of cell cycle and are thought to have a key function in the differentiation of various cell lines. We studied the effects of butyrate on differentiation, VDR expression, as well as on p21(Waf1/Cip1) and p27(Kip1) expression in human colon cancer cells (Caco-2). Butyrate induced cell differentiation, which was further enhanced after addition of 1,25-dihydroxycholecalciferol. Synergistic effect of butyrate and dihydroxycholecalciferol in Caco-2 cells was due to butyrate-induced overexpression of VDR. While butyrate as well as dihydroxycholecalciferol increased p21(Waf1/Cip1) and p27(Kip1) expression, in contrast combined exposure of butyrate and dihydroxycholecalciferol resulted in a synergistic amplification of p21(Waf1/Cip1), but not of p27(Kip1) expression. These data imply that butyrate selectively increases p21(Waf1/Cip1) expression via upregulation of VDR in Caco-2 cells.     

Pharmacological activation of cloned intermediate- and small-conductance Ca(2+)-activated K(+) channels

We previouslycharacterized 1-ethyl-2-benzimidazolinone (1-EBIO), as well as theclinically useful benzoxazoles, chlorzoxazone (CZ), and zoxazolamine(ZOX), as pharmacological activators of the intermediate-conductanceCa²⁺-activated K⁺ channel, hIK1. The mechanismof activation of hIK1, as well as the highly homologoussmall-conductance, Ca²⁺-dependent K⁺ channel,rSK2, was determined following heterologous expression inXenopus oocytes using two-electrode voltage clamp (TEVC) and excised, inside-out patch-clamp techniques. 1-EBIO, CZ, and ZOX activated both hIK1 and rSK2 in TEVC and excised inside-out patch-clamp experiments. In excised, inside-out patches, 1-EBIO and CZ induced aconcentration-dependent activation of hIK1, with half-maximal (K_1/2) values of 84 µM and 98 µM, respectively.Similarly, CZ activated rSK2 with a K_1/2 of 87 µM. In the absence of CZ, the Ca²⁺-dependent activationof hIK1 was best fit with a K_1/2 of 700 nM and aHill coefficient (n) of 2.0. rSK2 was activated byCa²⁺ with a K_1/2 of 700 nM and ann of 2.5. Addition of CZ had no effect on either theK_1/2 or n for Ca²⁺-dependentactivation of either hIK1 or rSK2. Rather, CZ increased channelactivity at all Ca²⁺ concentrations(V_max). Event-duration analysis revealed hIK1 wasminimally described by two open and three closed times. Activation by1-EBIO had no effect on _o1, _o2, or_c1, whereas _c2 and _c3 werereduced from 9.0 and 92.6 ms to 5.0 and 44.1 ms, respectively. Inconclusion, we define 1-EBIO, CZ, and ZOX as the first known activatorsof hIK1 and rSK2. Openers of IK and SK channels may be therapeuticallybeneficial in cystic fibrosis and vascular diseases.

     

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.

     

Modulation of cardiac Ca2+ channels by isoproterenol studied in transgenic mice with altered SR Ca2+ content

Phospholamban(PLB) ablation is associated with enhanced sarcoplasmic reticulum (SR)Ca²⁺ uptake and attenuation of thecardiac contractile responses to -adrenergic agonists. In thepresent study, we compared the effects of isoproterenol (Iso) on theCa²⁺ currents(I_Ca) ofventricular myocytes isolated from wild-type (WT) and PLB knockout(PLB-KO) mice. Current density and voltage dependence ofI_Ca were similarbetween WT and PLB-KO cells. However, I_Ca recorded fromPLB-KO myocytes had significantly faster decay kinetics. Iso increasedI_Ca amplitude inboth groups in a dose-dependent manner (50% effective concentration,57.1 nM). Iso did not alter the rate ofI_Ca inactivationin WT cells but significantly prolonged the rate of inactivation inPLB-KO cells. When Ba²⁺ was usedas the charge carrier, Iso slowed the decay of the current in both WTand PLB-KO cells. Depletion of SRCa²⁺ by ryanodine also slowed therate of inactivation ofI_Ca, and subsequent application of Iso further reduced the inactivation rate ofboth groups. These results suggest that enhancedCa²⁺ release from the SR offsetsthe slowing effects of -adrenergic receptor stimulation on the rateof inactivation ofI_Ca.

     

Temperature dependence of cloned mammalian and salmonid cardiac Na(+)/Ca(2+) exchanger isoforms

The cardiacNa⁺/Ca²⁺ exchanger (NCX), an importantregulator of cytosolic Ca²⁺ concentration in contractionand relaxation, has been shown in trout heart sarcolemmal vesicles tohave high activity at 7°C relative to its mammalian isoform. Thisunique property is likely due to differences in protein structure. Inthis study, outward NCX currents (I_NCX) of thewild-type trout (NCX-TR1.0) and canine (NCX 1.1) exchangers expressedin oocytes were measured to explore the potential contributions ofregulatory vs. transport mechanisms to this observation. cRNA wastranscribed in vitro from both wild-type cDNA and was injected intoXenopus oocytes. I_NCX of NCX-TR1.0 and NCX1.1 were measured after 3-4 days over a temperature range of 7-30°C using the giant excised patch technique. TheI_NCX for both isoforms exhibitedNa⁺-dependent inactivation and Ca²⁺-dependentpositive regulation. The I_NCX of NCX1.1exhibited typical mammalian temperature sensitivities withQ₁₀ values of 2.4 and 2.6 for peak and steady-statecurrents, respectively. However, the I_NCX ofNCX-TR1.0 was relatively temperature insensitive with Q₁₀values of 1.2 and 1.1 for peak and steady-state currents, respectively.I_NCX current decay was fit with a singleexponential, and the resultant rate constant of inactivation () wasdetermined as a function of temperature. As expected,  decreasedmonotonically with temperature for both isoforms. Although  wassignificantly greater in NCX1.1 compared with NCX-TR1.0 at alltemperatures, the effect of temperature on  was not differentbetween the two isoforms. These data suggest that thedisparities in I_NCX temperature dependencebetween these two exchanger isoforms are unlikely due to differences intheir inactivation kinetics. In addition, similar differences intemperature dependence were observed in both isoforms after-chymotrypsin treatment that renders the exchanger in a deregulatedstate. These data suggest that the differences in I_NCX temperature dependence between the twoisoforms are not due to potential disparities in either theI_NCX regulatory mechanisms or structuraldifferences in the cytoplasmic loop but are likely predicated ondifferences within the transmembrane segments.

     

Two-photon molecular excitation imaging of Ca2+ transients in Langendorff-perfused mouse hearts

The ability to image calciumsignals at subcellular levels within the intact depolarizing heartcould provide valuable information toward a more integratedunderstanding of cardiac function. Accordingly, a system combiningtwo-photon excitation with laser-scanning microscopy was developed tomonitor electrically evoked [Ca²⁺]_itransients in individual cardiomyocytes within noncontracting Langendorff-perfused mouse hearts. [Ca²⁺]_itransients were recorded at depths 100 µm from the epicardial surface with the fluorescent indicators rhod-2 or fura-2 in the presence of the excitation-contraction uncoupler cytochalasin D. Evoked[Ca²⁺]_i transients were highly synchronizedamong neighboring cardiomyocytes. At 1 Hz, the times from 90 to 50%(t_90-50%) and from 50 to 10%(t_50-10%) of the peak[Ca²⁺]_i were (means ± SE) 73 ± 4 and 126 ± 10 ms, respectively, and at 2 Hz, 62 ± 3 and94 ± 6 ms (n = 19, P < 0.05 vs.1 Hz) in rhod-2-loaded cardiomyocytes.[Ca²⁺]_i decay was markedly slower infura-2-loaded hearts (t_90-50% at 1 Hz,128 ± 9 ms and at 2 Hz, 88 ± 5 ms;t_50-10% at 1 Hz, 214 ± 18 ms and at2 Hz, 163 ± 7 ms; n = 19, P < 0.05 vs. rhod-2). Fura-2-induced deceleration of[Ca²⁺]_i decline resulted from increasedcytosolic Ca²⁺ buffering, because the kinetics of rhod-2decay resembled those obtained with fura-2 after incorporation of theCa²⁺ chelator BAPTA. Propagating calcium waves and[Ca²⁺]_i amplitude alternans were readilydetected in paced hearts. This approach should be of general utility tomonitor the consequences of genetic and/or functional heterogeneity incellular calcium signaling within whole mouse hearts at tissue depthsthat have been inaccessible to single-photon imaging.

     

Stimulation of unitary T-type Ca(2+) channel currents by calmodulin-dependent protein kinase II

The effect ofCa²⁺/calmodulin-dependent protein kinase II (CaMKII)stimulation on unitary low voltage-activated (LVA) T-type Ca²⁺ channel currents in isolated bovine adrenalglomerulosa (AG) cells was measured using the patch-clamp technique. Incell-attached and inside-out patches, LVA channel activity wasidentified by voltage-dependent inactivation and a single-channelconductance of ~9 pS in 110 mM BaCl₂ orCaCl₂. In the cell-attached patch, elevation of bathCa²⁺ from 150 nM to 1 µM raised intracellularCa²⁺ in K⁺-depolarized (140 mM) cells andevoked an increase in the LVA Ca²⁺ channel probability ofopening (NP_o) by two- to sixfold. This augmentation was associated with an increase in the number of nonblanksweeps, a rise in the frequency of channel opening in nonblank sweeps,and a 30% reduction in first latency. No apparent changes in thesingle-channel open-time distribution, burst lengths, or openings/burstwere apparent. Preincubation of AG cells with lipophilic or peptideinhibitors of CaMKII in the cell-attached or excised (inside-out)configurations prevented the rise in NP_o elicited by elevated Ca²⁺ concentration.Furthermore, administration of a mutant recombinant CaMKIIexhibiting cofactor-independent activity in the absence of elevatedCa²⁺ produced a threefold elevation in LVA channelNP_o. These data indicate that CaMKII activity isboth necessary and sufficient for LVA channel activation byCa²⁺.

     

Stimulation by caveolin-1 of the hypotonicity-induced release of taurine and ATP at basolateral, but not apical, membrane of Caco-2 cells

Regulatory volume decrease (RVD) is a protective mechanism that allows mammalian cells to restore their volume when exposed to a hypotonic environment. A key component of RVD is the release of K⁺, Cl^–, and organic osmolytes, such as taurine, which then drives osmotic water efflux. Previous experiments have indicated that caveolin-1, a coat protein of caveolae microdomains in the plasma membrane, promotes the swelling-induced Cl^– current (I_Cl,swell) through volume-regulated anion channels. However, it is not known whether the stimulation by caveolin-1 is restricted to the release of Cl^– or whether it also affects the swelling-induced release of other components, such as organic osmolytes. To address this problem, we have studied I_Cl,swell and the hypotonicity-induced release of taurine and ATP in wild-type Caco-2 cells that are caveolin-1 deficient and in stably transfected Caco-2 cells that express caveolin-1. Electrophysiological characterization of wild-type and stably transfected Caco-2 showed that caveolin-1 promoted I_Cl,swell, but not cystic fibrosis transmembrane conductance regulator currents. Furthermore, caveolin-1 expression stimulated the hypotonicity-induced release of taurine and ATP in stably transfected Caco-2 cells grown as a monolayer. Interestingly, the effect of caveolin-1 was polarized because only the release at the basolateral membrane, but not at the apical membrane, was increased. It is therefore concluded that caveolin-1 facilitates the hypotonicity-induced release of Cl^–, taurine, and ATP, and that in polarized epithelial cells, the effect of caveolin-1 is compartmentalized to the basolateral membrane. caveolae; osmolyte; epithelial cell; chloride channel     

Ca2+ depolarizes adrenal cortical cells through selective inhibition of an ATP-activated K+ current

Bovine adrenalzona fasciculata cells (AZF) express a noninactivatingK⁺ current(I_AC) whoseinhibition by adrenocorticotropic hormone and ANG II may be coupled tomembrane depolarization andCa²⁺-dependentcortisol secretion. We studiedI_ACinhibition byCa²⁺ and theCa²⁺ionophore ionomycin in whole cell and single-channel patch-clamp recordings of AZF. In whole cell recordings with intracellular (pipette)Ca²⁺concentration([Ca²⁺]_i)buffered to 0.02 µM,I_AC reachedmaximum current density of 25.0 ± 5.1 pA/pF(n = 16); raising[Ca²⁺]_ito 2.0 µM reduced it 76%. In inside-out patches, elevated[Ca²⁺]_idramatically reducedI_AC channelactivity. Ionomycin inhibited I_AC by 88 ± 4% (n = 14) without altering rapidlyinactivating A-type K⁺ current.Inhibition of I_ACby ionomycin was unaltered by adding calmodulin inhibitory peptide tothe pipette or replacing ATP with its nonhydrolyzable analog5'-adenylylimidodiphosphate.I_AC inhibition byionomycin was associated with membrane depolarization. When[Ca²⁺]_iwas buffered to 0.02 µM with 2 and 11 mM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ionomycin inhibitedI_AC by 89.6 ± 3.5 and 25.6 ± 14.6% and depolarized the same AZF by 47 ± 8 and 8 ± 3 mV, respectively (n = 4). ANG II inhibitedI_AC significantlymore effectively when pipette BAPTA was reduced from 11 to 2 mM. Raising[Ca²⁺]_iinhibits I_ACthrough a mechanism not requiring calmodulin or protein kinases,suggesting direct interaction withI_AC channels. ANGII may inhibitI_AC anddepolarize AZF by activating parallel signaling pathways, one of whichuses Ca²⁺ asa mediator.

     

L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

We investigatedthe role of intracellular calcium concentration([Ca²⁺]_i) in endothelin-1 (ET-1) production,the effects of potential vasospastic agents on[Ca²⁺]_i, and the presence of L-typevoltage-dependent Ca²⁺ channels in cerebral microvascularendothelial cells. Primary cultures of endothelial cells isolated frompiglet cerebral microvessels were used. Confluent cells were exposed toeither the thromboxane receptor agonist U-46619 (1 µM),5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca²⁺-chelatingagent EDTA (100 mM), the L-type Ca²⁺ channel blockerverapamil (10 µM), or the antagonist of receptor-operated Ca²⁺ channel SKF-96365 HCl (10 µM) for 15 min. ET-1production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT),or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. Toinvestigate the presence of L-type voltage-dependent Ca²⁺channels in endothelial cells, the [Ca²⁺]_isignal was determined fluorometrically by using fura 2-AM. Superfusionof confluent endothelial cells with U-46619, 5-HT, or LPA significantlyincreased [Ca²⁺]_i. Pretreatment ofendothelial cells with high K⁺ (60 mM) or nifedipine (4 µM) diminished increases in [Ca²⁺]_i inducedby the vasoactive agents. These results indicate that 1)elevated [Ca²⁺]_i signals are involved in ET-1biosynthesis induced by specific spasmogenic agents, 2) theincreases in [Ca²⁺]_i induced by thevasoactive agents tested involve receptor as well as L-typevoltage-dependent Ca²⁺ channels, and 3) primarycultures of cerebral microvascular endothelial cells express L-typevoltage-dependent Ca²⁺ channels.

     

H2O2 increases sheep tracheal blood flow, permeability, and vascular response to luminal capsaicin

Wells, U. M., S. Duneclift, and J. G. Widdicombe.H₂O₂increases sheep tracheal blood flow, permeability, and vascular response to luminal capsaicin. J. Appl.Physiol. 82(2): 621-631, 1997.Exogenous hydrogenperoxide(H₂O₂)causes airway epithelial damage in vitro. We have studied the effectsof luminalH₂O₂in the sheep trachea in vivo on tracheal permeability tolow-molecular-weight hydrophilic (technetium-99m-labeleddiethylenetriamine pentaacetic acid;^99mTc-DTPA) and lipophilic([¹⁴C]antipyrine;[¹⁴C]AP) tracers andon the tracheal vascular response to luminal capsaicin, whichstimulates afferent nerve endings. A tracheal artery was perfused, andtracheal venous blood was collected. H₂O₂exposure (10 mM) reduced tracheal potential difference(42.0 ± 6.4 mV) to zero. It increased arterial andvenous flows (56.7 ± 6.1 and 57.3 ± 10.0%,respectively; n = 5, P < 0.01, paired t-test) but not tracheal lymph flow(unstimulated flow 5.0 ± 1.2 µl ^· min^{1 ·} cm¹,n = 4). DuringH₂O₂exposure, permeability to ^99mTc-DTPA increased from2.6 to 89.7 × 10⁷ cm/s(n = 5, P < 0.05), whereas permeability to[¹⁴C]AP (3,312.6 × 10⁷ cm/s,n = 4) was not altered significantly(2,565 × 10⁷cm/s). Luminal capsaicin (10 µM) increased tracheal blood flow (10.1 ± 4.1%, n = 5)and decreased venous ^99mTc-DTPAconcentration (19.7 ± 4.0, P < 0.01), and these effects weresignificantly greater after epithelial damage (28.1 ± 6.0 and45.7 ± 4.3%, respectively,P < 0.05, unpairedt-test). Thus H₂O₂increases the penetration of a hydrophilic tracer into tracheal bloodand lymph but has less effect on a lipophilic tracer. It also enhancesthe effects of luminal capsaicin on blood flow and tracer uptake.

     

Role of aspartate residues in Ca(2+) affinity and permeation of the distal ECaC1

The Ca²⁺ affinity andpermeation of the epithelial Ca²⁺ channel (ECaC1) wereinvestigated after expression in Xenopus oocytes. ECaC1displayed anomalous mole-fraction effects. Extracellular Ca²⁺ and Mg²⁺ reversibly inhibited ECaC1 wholecell Li⁺ currents: IC₅₀ = 2.2 ± 0.4 µM (n = 9) and 235 ± 35 µM (n = 10), respectively. These values compare well with theCa²⁺ affinity of the L-type voltage-gated Ca²⁺(Ca_V1.2) channel measured under the same conditions,suggesting that high-affinity Ca²⁺ binding is awell-conserved feature of epithelial and voltage-gated Ca²⁺channels. Neutralization of D550 and E535 in the pore region had nosignificant effect on Ca²⁺ and Mg²⁺ affinities.In contrast, neutralization of D542 significantly decreasedCa²⁺ affinity (IC₅₀ = 1.1 ± 0.2 mM,n = 6) and Mg²⁺ affinity(IC₅₀ > 25 ± 3 mM, n = 4).Despite a 1,000-fold decrease in Ca²⁺ affinity in D542N,Ca²⁺ permeation properties and theCa²⁺-to-Ba²⁺ conductance ratio remainedcomparable to values for wild-type ECaC1. Together, our observationssuggest that D542 plays a critical role in Ca²⁺ affinitybut not in Ca²⁺ permeation in ECaC1.