Regulation of the mitochondrial permeability transition by matrix Ca(2+) and voltage during anoxia/reoxygenation

Westudied the interplay between matrix Ca²⁺ concentration([Ca²⁺]) and mitochondrial membrane potential() in regulation of the mitochondrial permeability transition(MPT) during anoxia and reoxygenation. Without Ca²⁺loading, anoxia caused near-synchronous dissipation,mitochondrial Ca²⁺ efflux, and matrix volume shrinkage whena critically low PO₂ was reached, which wasrapidly reversible upon reoxygenation. These changes were related toelectron transport inhibition, not MPT. Cyclosporin A-sensitive MPT didoccur when extramitochondrial [Ca²⁺] was increased topromote significant Ca²⁺ uptake during anoxia, depending onthe Ca²⁺ load size and ability to maintain . However,when [Ca²⁺] was increased after complete dissipation, MPT did not occur until reoxygenation, at which timereactivation of electron transport led to partial regeneration.In the setting of elevated extramitochondrial Ca²⁺, thisenhanced matrix Ca²⁺ uptake while promoting MPT because ofless than full recovery of . The interplay between andmatrix [Ca²⁺] in accelerating or inhibiting MPT duringanoxia/reoxygenation has implications for preventing reoxygenationinjury associated with MPT.

     

Na+ pump alpha 2-subunit expression modulates Ca2+ signaling

The role of the Na⁺ pump₂-subunit in Ca²⁺ signaling was examined inprimary cultured astrocytes from wild-type(₂^+/+ = WT) mouse fetuses and thosewith a null mutation in one [₂^+/ = heterozygote (Het)] or both [₂^/ = knockout (KO)] ₂ genes. Na⁺ pump catalytic() subunit expression was measured by immunoblot; cytosol[Na⁺] ([Na⁺]_cyt) and[Ca²⁺] ([Ca²⁺]_cyt) weremeasured with sodium-binding benzofuran isophthalate and fura 2 byusing digital imaging. Astrocytes express Na⁺ pumpswith both ₁- (80% of total ) and₂- (20% of total ) subunits. Het astrocytesexpress 50% of normal ₂; those from KO express none.Expression of ₁ is normal in both Het and KO cells.Resting [Na⁺]_cyt = 6.5 mM in WT, 6.8 mMin Het (P > 0.05 vs. WT), and 8.0 mM in KO cells(P < 0.001); 500 nM ouabain (inhibits only₂) equalized [Na⁺]_cyt at 8 mMin all three cell types. Resting[Ca²⁺]_cyt = 132 nM in WT, 162 nM in Het,and 196 nM in KO cells (both P < 0.001 vs. WT).Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER)Ca²⁺ pumps and unloads the ER, induces transient (inCa²⁺-free media) or sustained (in Ca²⁺-repletemedia) elevation of [Ca²⁺]_cyt. TheseCa²⁺ responses to 10 µM CPA were augmented in Het as wellas KO cells. When CPA was applied in Ca²⁺-free media, thereintroduction of Ca²⁺ induced significantly largertransient rises in [Ca²⁺]_cyt (due toCa²⁺ entry through store-operated channels) in Het and KOcells than in WT cells. These results correlate with published evidencethat ₂ Na⁺ pumps andNa⁺/Ca²⁺ exchangers are confined to plasmamembrane microdomains that overlie the ER. The data suggest thatselective reduction of ₂ Na⁺ pump activitycan elevate local [Na⁺] and, viaNa⁺/Ca²⁺ exchange, [Ca²⁺] in thetiny volume of cytosol between the plasma membrane and ER. This, inturn, augments adjacent ER Ca²⁺ stores and therebyamplifies Ca²⁺ signaling without elevating bulk[Na⁺]_cyt.

     

Impaired G(s)alpha and adenylyl cyclase cause beta-adrenoceptor desensitization in chronically hypoxic rat hearts

The effectsof -adrenoceptor stimulation with isoproterenol on electricallyinduced contraction and intracellular calcium ([Ca²⁺]_i) transient, and cAMP inmyocytes from both hypertrophied right and nonhypertrophied leftventricles of rats exposed to 10% oxygen for 4 wk, were significantlyattenuated. The increased [Ca²⁺]_i transientin response to cholera toxin was abolished, whereas increased cAMPafter NaF significantly attenuated. The biologically activeisoform, G_s-small (45 kDa), was reduced while thebiologically inactive isoform, G_s-large (52 kDa),increased. The increased electrically induced[Ca²⁺]_i transient and cAMP with 10-100µM forskolin were significantly attenuated in chronically hypoxicrats. The content of G_i2, the predominantisoform of G_i protein in the heart, was unchanged. Resultsindicate that impaired functions of G_s protein and adenylyl cyclase cause -adrenoceptor desensitization. The impaired function of the G_s protein may be due to reducedG_s-small and/or increased G_s-large, whichdoes not result from changes in G_i protein. Responses toall treatments were the same for right and left ventricles, indicatingthat the impaired cardiac functions are not secondary to cardiac hypertrophy.

     

Carbachol-induced desensitization of PLC-beta pathway in rat myometrium: downregulation of Gqalpha /G11alpha

In the estrogen-treated rat myometrium, carbachol increased thegeneration of inositol phosphates by stimulating the muscarinic receptor-G_q/G₁₁-phospholipaseC-3 (PLC-3) cascade. Exposure to carbachol resulted in a rapidand specific (homologous) attenuation of the subsequent muscarinicresponses in terms of inositol phosphate production, PLC-3translocation to membrane, and contraction. Refractoriness wasaccompanied by a reduction of membrane muscarinic binding sites and anuncoupled state of residual receptors. Protein kinase C (PKC) alteredthe functionality of muscarinic receptors and contributed to theinitial period of desensitization. A delayed phase of the muscarinicrefractoriness was PKC independent and was associated with adownregulation ofG_q/G₁₁.Atropine failed to induce desensitization as well asG_q/G₁₁downregulation, indicating that both events involve active occupancy ofthe receptor. Prolonged exposure toAlF₄ reduced subsequent AlF₄ as well as carbachol-mediatedinositol phosphate responses and similarly induced downregulation ofG_q/G₁₁. Data suggest that a decrease in the level ofG_q/G₁₁is subsequent to its activation and may account forheterologous desensitization.

     

Polyamines regulate beta-catenin tyrosine phosphorylation via Ca(2+) during intestinal epithelial cell migration

Polyaminesare essential for early mucosal restitution that occurs by epithelialcell migration to reseal superficial wounds after injury. Normalintestinal epithelial cells are tightly bound in sheets, but they needto be rapidly disassembled during restitution. -Catenin is involvedin cell-cell adhesion, and its tyrosine phosphorylation causesdisassembly of adhesion junctions, enhancing the spreading of cells.The current study determined whether polyamines are required for thestimulation of epithelial cell migration by altering -catenintyrosine phosphorylation. Migration of intestinal epithelial cells(IEC-6 line) after wounding was associated with an increase in-catenin tyrosine phosphorylation, which decreased the bindingactivity of -catenin to -catenin. Polyamine depletion by-difluoromethylornithine reduced cytoplasmic free Ca²⁺concentration ([Ca²⁺]_cyt), preventedinduction of -catenin phosphorylation, and decreased cell migration.Elevation of [Ca²⁺]_cyt induced by theCa²⁺ ionophore ionomycin restored -cateninphosphorylation and promoted migration in polyamine-deficient cells.Decreased -catenin phosphorylation through the tyrosine kinaseinhibitor herbimycin-A or genistein blocked cell migration, which wasaccompanied by reorganization of cytoskeletal proteins. These resultsindicate that -catenin tyrosine phosphorylation plays a criticalrole in polyamine-dependent cell migration and that polyamines induce-catenin tyrosine phosphorylation at least partially through[Ca²⁺]_cyt.

     

Synergism between toxin-gamma from Brazilian scorpion Tityus serrulatus and veratridine in chromaffin cells

Toxin- (T)from the Brazilian scorpion Tityusserrulatus venom caused a concentration- andtime-dependent increase in the release of norepinephrine andepinephrine from bovine adrenal medullary chromaffin cells. T was~200-fold more potent than veratridine judged fromEC₅₀ values, although the maximalsecretory efficacy of veratridine was 10-fold greater than that of T(1.2 vs. 12 µg/ml of catecholamine release). The combination of both toxins produced a synergistic effect that was particularly drastic at 5 mM extracellular Ca²⁺concentration([Ca²⁺]_o),when 30 µM veratridine plus 0.45 µM T were used. T (0.45 µM) doubled the basal uptake of⁴⁵Ca²⁺,whereas veratridine (100 µM) tripled it. Again, a drastic synergism in enhancing Ca²⁺ entry was seenwhen T and veratridine were combined; this was particularlypronounced at 5 mM[Ca²⁺]_o.Veratridine induced oscillations of cytosolicCa²⁺ concentration([Ca²⁺]_i)in single fura 2-loaded cells without elevation of basal levels. Incontrast, T elevated basal[Ca²⁺]_ilevels, causing only small oscillations. When added together, T andveratridine elevated the basal levels of[Ca²⁺]_iwithout causing large oscillations. T shifted the current-voltage (I-V) curve forNa⁺ channel current to the left.The combination of T with veratridine increased the shift of theI-V curve to the left, resulting in agreater recruitment of Na⁺channels at more hyperpolarizing potentials. This led to enhanced andmore rapid accumulation of Na⁺ inthe cell, causing cell depolarization, the opening of voltage-dependent Ca²⁺ channels, andCa²⁺ entry and secretion.

     

Excitation wavelengths for fura 2 provide a linear relationship between [Ca(2+)] and fluorescence ratio

We proposed andtested the use of nontraditional excitation wavelengths(₁ and ₂) and an emission wavelength(_em) to define conditions under which free calciumconcentration and a fluorescence ratio are linearly related.Fluorescence spectra were determined for aqueous solutions thatcontained 25 µM fura 2, 125 mM K⁺, and either 0 mM or 0.1 mM Ca²⁺. Effectively linear relationships between[Ca²⁺] and a fluorescence ratio, i.e., <5% bias when[Ca²⁺]  5 × dissociation constant, were apparentwhen ₁  400 nm, ₂  370 nm, and_em  510 nm. Combinations with longer ₁and _em and/or with shorter ₂ reduced thisbias further. Although the method described does not obviate thecomplications that surround the correction for fluorescence background,choosing a nontraditional combination of excitation and emissionwavelengths offers several practical advantages over more traditionalfura 2 fluorescence methodologies in a variety of experimental settings.

     

Heterologous desensitization of response mediated by selective PKC-dependent phosphorylation of G(i-1) and G(i-2)

This study examined the ability of protein kinase C (PKC) toinduce heterologous desensitization by targeting specific G proteinsand limiting their ability to transduce signals in smooth muscle.Activation of PKC by pretreatment of intestinal smooth muscle cellswith phorbol 12-myristate 13-acetate, cholecystokinin octapeptide, orthe phosphatase 1 and phosphatase 2A inhibitor, calyculin A,selectively phosphorylated G_i-1 and G_i-2,but not G_i-3 or G_o, and blockedinhibition of adenylyl cyclase mediated by somatostatin receptorscoupled to G_i-1 and opioid receptors coupled toG_i-2, but not by muscarinic M₂ and adenosineA₁ receptors coupled to G_i-3. Phosphorylationof G_i-1 and G_i-2 and blockade of cyclaseinhibition were reversed by calphostin C and bisindolylmaleimide, andadditively by selective inhibitors of PKC and PKC. Blockade ofinhibition was prevented by downregulation of PKC. Phosphorylation ofG-subunits by PKC also affected responses mediated by-subunits. Pretreatment of muscle cells withcANP-(4-23), a selective agonist of the natriureticpeptide clearance receptor, NPR-C, which activates phospholipase C(PLC)-3 via the -subunits of G_i-1 andG_i-2, inhibited the PLC- response to somatostatin and[D-Pen^2,5]enkephalin. The inhibition waspartly reversed by calphostin C. Short-term activation of PKC had noeffect on receptor binding or effector enzyme (adenylyl cyclase orPLC-) activity. We conclude that selective phosphorylation ofG_i-1 and G_i-2 by PKC partly accounts forheterologous desensitization of responses mediated by the - and-subunits of both G proteins. The desensitization reflects adecrease in reassociation and thus availability of heterotrimeric G proteins.

     

Arachidonic acid mediates dual effect of TNF-alpha on Ca2+ transients and contraction of adult rat cardiomyocytes

Tumor necrosisfactor (TNF)- has a biphasic effect on heart contractility andstimulates phospholipase A₂ (PLA₂) incardiomyocytes. Because arachidonic acid (AA) exerts a dual effect onintracellular Ca²⁺ concentration([Ca²⁺]_i) transients, we investigated thepossible role of AA as a mediator of TNF- on[Ca²⁺]_i transients and contraction withelectrically stimulated adult rat cardiac myocytes. At a lowconcentration (10 ng/ml) TNF- produced a 40% increase in theamplitude of both [Ca²⁺]_i transients andcontraction within 40 min. At a high concentration (50 ng/ml) TNF-evoked a biphasic effect comprising an initial positive effect peakingat 5 min, followed by a sustained negative effect leading to50-40% decreases in [Ca²⁺]_i transientsand contraction after 30 min. Both the positive and negative effects ofTNF- were reproduced by AA and blocked by arachidonyltrifluoromethylketone (AACOCF3), an inhibitor of cytosolic PLA₂.Lipoxygenase and cyclooxygenase inhibitors reproduced the high-doseeffects of TNF- and AA. The negative effects of TNF- and AA werealso reproduced by sphingosine and were abrogated by the ceramidaseinhibitor n-oleoylethanolamine. These results point out thekey role of the cytosolic PLA₂/AA pathway in mediating thecontractile effects of TNF-.

     

Intracellular H+ regulates the alpha -subunit of ENaC, the epithelial Na+ channel

Protons regulateelectrogenic sodium absorption in a variety of epithelia, including thecortical collecting duct, frog skin, and urinary bladder. Recently,three subunits (, , ) coding for the epithelial sodium channel(ENaC) were cloned. However, it is not known whether pH regulatesNa⁺ channels directly byinteracting with one of the three ENaC subunits or indirectly byinteracting with a regulatory protein. As a first step to identifyingthe molecular mechanisms of proton-mediated regulation of apicalmembrane Na⁺ permeability inepithelia, we examined the effect of pH on the biophysical propertiesof ENaC. To this end, we expressed various combinations of -, -,and -subunits of ENaC in Xenopusoocytes and studied ENaC currents by the two-electrode voltage-clampand patch-clamp techniques. In addition, the effect of pH on the-ENaC subunit was examined in planar lipid bilayers. We report that ,,-ENaC currents were regulated by changes in intracellular pH(pH_i) but not by changes inextracellular pH (pH_o).Acidification reduced and alkalization increased channel activity by avoltage-independent mechanism. Moreover, a reduction ofpH_i reduced single-channel openprobability, reduced single-channel open time, and increased single-channel closed time without altering single-channel conductance. Acidification of the cytoplasmic solution also inhibited ,-ENaC, ,-ENaC, and -ENaC currents. We conclude thatpH_i but notpH_o regulates ENaC and that the-ENaC subunit is regulated directly bypH_i.     

TGF-alpha reduces bradykinin-stimulated ion transport and prostaglandin release in human colonic epithelial cells

The effect of chronic exposure to transforming growth factor-(TGF-) on bradykinin-stimulated acute prostanoid production and ionsecretion in monolayers of HCA-7 colony 29 colonic epithelial cells hasbeen studied. Monolayers synthesized prostaglandinE₂ (PGE₂) at a basal rate of 2.10 ± 0.31 pg · monolayer¹ · min¹over 24 h. Bradykinin(10⁸-10⁵M) dose dependently increased acutePGE₂ release by three orders ofmagnitude. This was associated with a rise in cAMP from 1.60 ± 0.14 to 2.90 ± 0.1 pmol/monolayer (P < 0.02) and a dose-dependent increase in short-circuit current (SCC).When monolayers were primed by a 24-h exposure to TGF-, basalPGE₂ release rose to 6.31 ± 0.38 pg · monolayer¹ · min¹(TGF- concn 10 ng/ml; P = 0.001).However, the stimulation of acute prostaglandin release, intracellularcAMP, and increased SCC by bradykinin was significantly reduced bypreincubation with TGF-. Priming withPGE₂(10⁸-10⁶M) over 24 h mimicked the effect of TGF- on bradykinin-induced changes in cAMP and SCC. These data suggest that enhanced chronic release of prostaglandins in response to stimulation with TGF- maydownregulate acute responses to bradykinin. In vivo, TGF- could havean important modulatory function in regulating secretion underinflammatory conditions.     

PKA and arachidonic acid activation of human recombinant ClC-2 chloride channels

An HEK-293 cell line stably expressing the humanrecombinant ClC-2 Cl channel was used in patch-clampstudies to study its regulation. The relative permeabilityP_x/P_Cl calculated fromreversal potentials was I > Cl = NO₃ = SCNBr. Theabsolute permeability calculated from conductance ratios wasCl = Br = NO₃  SCN > I. The channel was activatedby cAMP-dependent protein kinase (PKA), reduced extracellular pH, oleicacid (C:18 cis9), elaidic acid (C:18trans9), arachidonic acid (AA; C:20cis5,8,11,14), and by inhibitors of AA metabolism,5,8,11,14-eicosatetraynoic acid (ETYA; C:20trans5,8,11,14),-methyl-4-(2-methylpropyl)benzeneacetic acid (ibuprofen), and2-phenyl-1,2-benzisoselenazol-3-[2H]-one (PZ51, ebselen). ClC-2Cl channels were activated by a combination of forskolinplus IBMX and were inhibited by the cell-permeant myristoylated PKAinhibitor (mPKI). Channel activation by reduction of bath pH wasincreased by PKA and prevented by mPKI. AA activation of the ClC-2Cl channel was not inhibited by mPKI or staurosporine andwas therefore independent of PKA or protein kinase C activation.

     

Modulation of human neuronal alpha 1E-type calcium channel by alpha 2delta -subunit

Calcium channels are composed of a pore-forming subunit,₁, and at least two auxiliarysubunits, - and₂-subunits. It is well knownthat -subunits regulate most of the properties of the channel. Thefunction of ₂-subunit isless understood. In this study, the effects of the calcium channel₂-subunit on the neuronal_1E voltage-gated calciumchannel expressed in Xenopus oocyteswas investigated without and with simultaneous coexpression of eitherthe _1b- or the_2a-subunit. Most aspects of_1E function were affected by₂. Thus₂ caused a shift in thecurrent-voltage and conductance-voltage curves toward more positivepotentials and accelerated activation, deactivation, and theinstallation of the inactivation process. In addition, the efficiencywith which charge movement is coupled to pore opening assessed bydetermining ratios of limiting conductance to limiting charge movementwas decreased by ₂ byfactors that ranged from 1.6 (P < 0.01) for _1E-channels to 3.0 (P < 0.005) for_1E1b-channels. These results indicate that₂ facilitates the expressionand the maturation of_1E-channels and converts thesechannels into molecules responding more rapidly to voltage.

     

Differences in Ca(2+) signaling underlie age-specific effects of secretagogues on colonic Cl(-) transport

Taurodeoxycholic acid (TDC) stimulates Cl transport inadult (AD), but not weanling (WN) and newborn (NB), rabbit colonic epithelial cells (colonocytes). The present study demonstrates thatstimuli like neurotensin (NT) are also age specific and identifies theage-dependent signaling step. Bile acid actions are segment and bileacid specific. Thus although TDC and taurochenodeoxycholate stimulateCl transport in AD distal but not proximal colon,taurocholate has no effect in either segment. TDC increasesintracellular Ca²⁺ concentration([Ca²⁺]_i) in AD, but not in WN and NB,colonocytes. In AD cells, TDC (5 min) action on Cltransport needs intra- but not extracellular Ca²⁺. NT,histamine, and bethanechol increase Cl transport and[Ca²⁺]_i in AD, but not WN, distalcolonocytes. However, A-23187 increased [Ca²⁺]_i and Cl transport in allage groups, suggesting that Ca²⁺-sensitive Cltransport is present from birth. Study of the proximal steps inCa²⁺ signaling revealed that NT, but not TDC, activates aGTP-binding protein, G_q, in AD and WN cells. Inaddition, although WN and AD colonocytes had similar levels ofphosphatidylinositol 4,5-bisphosphate, NT and TDC increased1,4,5-inositol trisphosphate content only in AD cells.Nonresponsiveness of WN cells to Ca²⁺-dependent stimuli,therefore, is due to the absence of measurable phospholipase Cactivity. Thus delays in Ca²⁺ signaling afford a crucialprotective mechanism to meet the changing demands of the developing colon.

     

IP3 receptor blockade fails to prevent intracellular Ca2+ release by ET-1 and alpha -thrombin

The effect ofinositol 1,4,5-trisphosphate(IP₃) receptor blockade onplatelet-derived growth factor (PDGF), fibroblast growth factor (FGF),endothelin-1 (ET-1), or -thrombin receptor-mediated intracellularCa²⁺(Ca²⁺_i) release was examined using fura 2 microspectrofluorometry in single Chinese hamster ovary cells andmyoblasts. Blockade of the IP₃receptor was achieved by microinjection of heparin or monoclonalantibody (MAb) 18A10 into the IP₃type 1 receptor. Heparin completely inhibitedCa²⁺_i release after flash photolysis withcaged IP₃ and after exposure toPDGF and FGF. In contrast, heparin failed to blockCa²⁺_i release after -thrombin andET-1. After application of ligand, IP₃ levels were five- to sevenfoldhigher for -thrombin than for ET-1 or PDGF.IP₃ levels after PDGF and ET-1were comparable. Similar to heparin, MAb 18A10 blockedCa²⁺_i release after PDGF but failed toblock Ca²⁺_i release after ET-1 or-thrombin. These data suggest that the mechanisms of Ca²⁺_i release by tyrosine kinase andcertain 7-transmembrane receptors may differ. Although both receptortypes use the IP₃-signaling system, the ET-1 and -thrombin receptors may have a second,alternative mechanism for activatingCa²⁺_i release.

     

beta-adrenergic receptor/cAMP-mediated signaling and apoptosis of S49 lymphoma cells

-Adrenergic receptor (AR) activationand/or increases in cAMP regulate growth and proliferation of a varietyof cells and, in some cells, promote cell death. In the current studieswe addressed the mechanism of this growth reduction by examiningAR-mediated effects in the murine T-lymphoma cell line S49.Wild-type S49 cells, derived from immature thymocytes(CD4⁺/CD8⁺) undergo growth arrest andsubsequent death when treated with agents that increase cAMP levels(e.g., AR agonists, 8-bromo-cAMP, cholera toxin, forskolin).Morphological and biochemical criteria indicate that this cell death isa result of apoptosis. In cyc and kin S49cells, which lack G_s and functional protein kinase A(PKA), respectively, AR activation of G_s and cAMPaction via PKA are critical steps in this apoptotic pathway. S49 cellsthat overexpress Bcl-2 are resistant to cAMP-induced apoptosis. Weconclude that AR activation induces apoptosis in immature Tlymphocytes via G_s and PKA, while overexpression ofBcl-2 prevents cell death. AR/cAMP/PKA-mediated apoptosis mayprovide a means to control proliferation of immature T cells in vivo.

     

Permeant but not impermeant divalent cations enhance activation of nondesensitizing alpha(7) nicotinic receptors

Neuronal₇ nicotinic acetylcholine receptors (nAChRs) arepermeable to Ca²⁺ and other divalent cations. Wecharacterized the modulation of the pharmacological properties ofnondesensitizing mutant (L²⁴⁷T andS²⁴⁰T/L²⁴⁷T) ₇ nAChRs bypermeant (Ca²⁺, Ba²⁺, and Sr²⁺) andimpermeant (Cd²⁺ and Zn²⁺) divalent cations.₇ receptors were expressed in Xenopus oocytes and studied with two-electrode voltage clamp. Extracellular permeant divalent cations increased the potency and maximal efficacy of ACh,whereas impermeant divalent cations decreased potency and maximalefficacy. The antagonist dihydro--erythroidine (DHE) was a strongpartial agonist of L²⁴⁷T andS²⁴⁰T/L²⁴⁷T ₇ receptors in thepresence of divalent cations but was a weak partial agonist in thepresence of impermeant divalent cations. Mutation of the"intermediate ring" glutamates (E²³⁷A) inL²⁴⁷T ₇ nAChRs eliminated Ca²⁺conductance but did not alter the Ca²⁺-dependent increasein ACh potency, suggesting that site(s) required for modulation are onthe extracellular side of the intermediate ring. The difference betweenpermeant and impermeant divalent cations suggests that sites within thepore are important for modulation by divalent cations.

     

Ouabain and substrate affinities of human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) when expressed separately in yeast cells

HumanNa⁺-K⁺-ATPase₁₁,₂₁, and ₃₁heterodimers were expressed individually in yeast, and ouabainbinding and ATP hydrolysis were measured in membrane fractions. Theouabain equilibrium dissociation constant was 13-17 nM for₁₁ and ₃₁at 37°C and 32 nM for ₂₁, indicatingthat the human -subunit isoforms have a similar high affinity forcardiac glycosides. K_0.5 values for antagonism of ouabain binding by K⁺ were ranked in order as follows:₂ (6.3 ± 2.4 mM) > ₃(1.6 ± 0.5 mM)  ₁ (0.9 ± 0.6 mM),and K_0.5 values for Na⁺ antagonismof ouabain binding to all heterodimers were 9.5-13.8 mM. Themolecular turnover for ATP hydrolysis by₁₁ (6,652 min¹) was abouttwice as high as that by ₃₁ (3,145 min¹). These properties of the human heterodimersexpressed in yeast are in good agreement with properties of the humanNa⁺-K⁺-ATPase expressed in Xenopusoocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-DHorisberger, L Lelievie, and K Geering. J Biol Chem275: 1976-1986, 2000). In contrast to Na⁺ pumpsexpressed in Xenopus oocytes, the₂₁ complex in yeast membranes wassignificantly less stable than ₁₁ or₃₁, resulting in a lower functionalexpression level. The ₂₁ complex was also more easily denatured by SDS than was the₁₁ or the₃₁ complex.

     

Protein kinase Cbeta regulates heterologous desensitization of thrombin receptor (PAR-1) in endothelial cells

We studied the effects of protein kinase C (PKC) activation onendothelial cell surface expression and function of the proteolytically activated thrombin receptor 1 (PAR-1). Cell surface PAR-1 expression was assessed by immunofluorescence (using anti-PAR-1 monoclonal antibody), and receptor activation was assessed by measuring increases in cytosolic Ca²⁺ concentration inhuman dermal microvascular endothelial cells (HMEC) exposed to-thrombin or phorbol ester,12-O-tetradecanoylphorbol-13-acetate (TPA).Immunofluorescence showed that thrombin and TPA reduced the cellsurface expression of PAR-1. Prior exposure of HMEC to thrombin for 5 min desensitized the cells to thrombin, indicating homologous PAR-1desensitization. In contrast, prior activation of PKC with TPA produceddesensitization to thrombin and histamine, indicatingheterologous PAR-1 desensitization. Treatment of cells withstaurosporine, a PKC inhibitor, fully prevented heterologous desensitization, whereas thrombin-induced homologous desensitization persisted. Depletion of PKC isozymes(PKC_I andPKC_II) by transducing cellswith antisense cDNA of PKC_Iprevented the TPA-induced decrease in cell surface PAR-1 expression andrestored ~60% of the cytosolic Ca²⁺ signal in response tothrombin. In contrast, depletion of PKC isozymes did not affect theloss of cell surface PAR-1 and induction of homologous PAR-1desensitization by thrombin. Therefore, homologous PAR-1desensitization by thrombin occurs independently of PKC isozymes,whereas the PKC-activated pathway is important in signaling heterologous PAR-1 desensitization in endothelial cells.

     

Agonist-specific cross talk between ERKs and p38(mapk) regulates PGI(2) synthesis in endothelium

We have examined the mechanisms regulatingprostacyclin (PGI₂) synthesis after acute exposure of humanumbilical vein endothelial cells (HUVEC) to interleukin-1 (IL-1).IL-1 evoked an early (30 min) release of PGI₂ and[³H]arachidonate that was blocked by the cytosolicphospholipase A₂ (cPLA₂) inhibitorarachidonyl trifluoromethyl ketone. IL-1-mediated activationof extracellular signal-regulated kinase 1/2 (ERK1/2; p42/p44^mapk) coincided temporally with phosphorylation ofcPLA₂ and with the onset of PGI₂synthesis. The mitogen-activated protein kinase (MAPK) kinase (MEK)inhibitors, PD-98059 and U-0126, blocked IL-1-induced ERKactivation and partially attenuated cPLA₂phosphorylation and PGI₂ release, suggesting thatERK-dependent and -independent pathways regulate cPLA₂phosphorylation. SB-203580 treatment enhanced IL-1-induced MEK,p42/44^mapk, and cPLA₂ phosphorylation butreduced thrombin-stimulated MEK and p42/44^mapk activation.IL-1, but not thrombin, activated Raf-1 as assessed byimmune-complex kinase assay, as did SB-203580 alone. These results showthat IL-1 causes an acute upregulation of PGI₂generation in HUVEC, establish a role for theMEK/ERK/cPLA₂ pathway in this early release, and provideevidence for an agonist-specific cross talk between p38^mapkand p42/44^mapk that may reflect receptor-specificdifferences in the signaling elements proximal to MAPK activation.