Antisense oligodeoxynucleotide to PKC-delta blocks alpha 1-adrenergic activation of Na-K-2Cl cotransport

A role for protein kinase C (PKC)- and -isotypes in ₁-adrenergicregulation of human tracheal epithelial Na-K-2Cl cotransport wasstudied with the use of isotype-specific PKC inhibitors and antisenseoligodeoxynucleotides to PKC- or - mRNA. Rottlerin, a PKC-inhibitor, blocked 72% of basolateral-to-apical, bumetanide-sensitive ³⁶Cl flux innystatin-permeabilized cell monolayers stimulated with methoxamine, an₁-adrenergic agonist, with a50% inhibitory concentration of 2.3 µM. Methoxamine increased PKCactivity in cytosol and a particulate fraction; the response wasinsensitive to PKC- and -_IIisotype-specific inhibitors, but was blocked by general PKC inhibitorsand rottlerin. Rottlerin also inhibited methoxamine-induced PKCactivity in immune complexes of PKC-, but not PKC-. At the subcellular level, methoxamine selectively elevated cytosolic PKC-activity and particulate PKC- activity. Pretreatment of cellmonolayers with antisense oligodeoxynucleotide to PKC- for 48 hreduced the amount of whole cell and cytosolic PKC-, diminished whole cell and cytosolic PKC- activity, and blockedmethoxamine-stimulated Na-K-2Cl cotransport. Sense oligodeoxynucleotideto PKC- and antisense oligodeoxynucleotide to PKC- did not altermethoxamine-induced cotransport activity. These results demonstrate theselective activation of Na-K-2Cl cotransport by cytosolic PKC-.

     

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.

     

Serine phosphorylation of p60 tumor necrosis factor receptor by PKC-delta in TNF-alpha-activated neutrophils

Tumor necrosis factor-(TNF-) triggers degranulation and oxygen radical release in adherentneutrophils. The p60TNF receptor (p60TNFR) is responsible forproinflammatory signaling, and protein kinase C (PKC) is a candidatefor the regulation of p60TNFR. Both TNF- and the PKC-activatorphorbol 12-myristate 13-acetate triggered phosphorylation of p60TNFR.Receptor phosphorylation was on both serine and threonine but not ontyrosine residues. The PKC- isotype is a candidate enzyme for serinephosphorylation of p60TNFR. Staurosporine and the PKC- inhibitorrottlerin inhibited TNF--triggered serine but not threoninephosphorylation. Serine phosphorylation was associated withreceptor desensitization, as inhibition of PKC resulted in enhanceddegranulation (elastase release). After neutrophil activation, PKC-was the only PKC isotype that associated with p60TNFR within thecorrect time frame for receptor phosphorylation. In vitro, onlyPKC-, but not the -, I-, II-, or -isotypes, wascompetent to phosphorylate the receptor, indicating that p60TNFR is adirect substrate for PKC-. These findings suggest a selective rolefor PKC- in negative regulation of the p60TNFR and ofTNF--induced signaling.

     

Antisense oligonucleotide to PKC-epsilon alters cAMP-dependent stimulation of CFTR in Calu-3 cells

Protein kinase C(PKC) regulates cystic fibrosis transmembrane conductance regulator(CFTR) channel activity but the PKC signaling mechanism is not yetknown. The goal of these studies was to identify PKC isotype(s)required for control of CFTR function. CFTR activity was measured as³⁶Cl efflux in a Chinese hamsterovary cell line stably expressing wild-type CFTR (CHO-wtCFTR) and in aCalu-3 cell line. Chelerythrine, a PKC inhibitor, delayed increasedCFTR activity induced with phorbol 12-myristate 13-acetate or with thecAMP-generating agents ()-epinephrine or forskolin plus8-(4-chlorophenylthio)adenosine 3',5'- cyclicmonophosphate. Immunoblot analysis of Calu-3 cells revealed thatPKC-, -_II, -, -, and- were expressed in confluent cell cultures. Pretreatment of cellmonolayers with Lipofectin plus antisense oligonucleotide to PKC-for 48 h prevented stimulation of CFTR with ()-epinephrine,reduced PKC- activity in unstimulated cells by 52.1%, and decreasedPKC- mass by 76.1% but did not affect hormone-activated proteinkinase A activity. Sense oligonucleotide to PKC- and antisenseoligonucleotide to PKC- and - did not alter()-epinephrine-stimulated CFTR activity. These results demonstrate the selective regulation of CFTR function by constitutively active PKC-.

     

Differential translocation of protein kinase C isozymes by phorbol esters, EGF, and ANG II in rat liver WB cells

The protein kinaseC (PKC) family represents an important group of enzymes whoseactivation is associated with their translocation from the cytosol todifferent cellular membranes. In this study, the spatial distributionof PKC-, - and - in rat liver epithelial (WB) cells has beenexamined by Western blot analysis after subcellular fractionation.Cytosolic, membrane, nuclear, and cytoskeletal fractions were obtainedfrom cells stimulated with phorbol 12-myristate 13-acetate (PMA),angiotensin II (ANG II), or epidermal growth factor (EGF). PMA causedmost of the PKC-, - and - initially present in the cytosol tobe transported to the membrane and nuclear fractions. In contrast, bothANG II and EGF induced only a minor translocation of PKC- to themembrane fraction but caused a statistically significantmembrane-directed movement of PKC- and -. Translocation ofPKC- and - to the nucleus induced by ANG II and EGF was transient and quantitatively smaller than that induced by PMA. PKC- and -were present in the cytoskeleton of resting cells, but although PMA,ANG II, and EGF caused some changes in their content, these werevariable, suggesting that the cytoskeleton fraction was heterogeneous. PKC depletion inhibited ANG II-induced mitogenesis and the sustained activation of Raf-1 and extracellular regulated protein kinase (ERK).However, although PKC depletion inhibited EGF-induced mitogenesis, themaximum EGF-induced activation of the ERK pathway was only slightlyretarded. We hypothesize that PKC- and - are involved inmitogenesis via both ERK-dependent and ERK-independent mechanisms. These results support the notion that specific PKC isozymes exert spatially defined effects by virtue of their directed translocation todistinct intracellular sites.

     

Dopamine-induced translocation of protein kinase C isoforms visualized in renal epithelial cells

Short-term regulation of sodiummetabolism is dependent on the modulation of the activity of sodiumtransporters by first and second messengers. In understanding diseasesassociated with sodium retention, it is necessary to identify thecoupling between these messengers. We have examined whether dopamine,an important first messenger in tubular cells, activates andtranslocates various protein kinase C (PKC) isoforms. We used aproximal tubular-like cell line, LLCPK-1 cells, in which dopamine wasfound to inhibit Na⁺-K⁺-ATPase in aPKC-dependent manner. Translocation of PKC isoforms was studied withboth subcellular fractionation and confocal microscopy. Both techniquesrevealed a dopamine-induced translocation from cytosol to plasmamembrane of PKC- and -, but not of PKC-, -, and -. Theprocess of subcellular fractionation resulted in partial translocationof PKC-. This artifact was eliminated in confocal studies. Confocalimaging permitted detection of translocation within 20 s.Translocation was abolished by a phospholipase C inhibitor and by anantagonist against the dopamine 1 subtype (D₁) but not the2 subtype of receptor (D₂). In conclusion, this studyvisualizes in renal epithelial cells a very rapid activation of thePKC- and - isoforms by the D₁ receptor subtype.

     

Role of beta 1- and beta 3-adrenoceptors in the regulation of lipolysis and thermogenesis in rat brown adipocytes

To evaluate the physiological functions of₁-,₂-, and₃-adrenoceptors (ARs) in brownadipose tissue, the lipolytic and respiratory effects of variousadrenergic agonists and antagonists were studied in rat brownadipocytes. The -agonists stimulated both lipolysis and respiration(8-10 times above basal levels), with the following order ofpotency (concentration eliciting 50% of maximum response):CL-316243 (₃) > BRL-37344(₃) > isoproterenol (mainly₁/₂) > norepinephrine (NE; mainly₁/₂) > epinephrine (mainly₁/₂) dobutamine (₁)  procaterol (₂). Schild plot coefficients of competitive inhibition experiments using ICI-89406 (₁ antagonist) revealed thatmore than one type of receptor mediates NE action. It is concluded fromour results that 1) NE, at low plasma levels (1-25 nM), stimulates lipolysis and respiration mainly through ₁-ARs,2) NE, at higher levels, stimulateslipolysis and respiration via both₁- and₃-ARs,3)₂-ARs play only a minor role,and 4)₃-ARs may represent thephysiological receptors for the high NE concentrations in the synapticcleft, where the high-affinity₁-ARs are presumablydesensitized. It is also suggested that lipolysis represents theflux-generating step regulating mitochondrial respiration.

     

Stability of actin cytoskeleton and PKC-delta binding to actin regulate NKCC1 function in airway epithelial cells

Activation of airwayepithelial Na-K-2Cl cotransporter (NKCC)1 requires increased activityof protein kinase C (PKC)-, which localizes predominantly to theactin cytoskeleton. Prompted by reports of a role for actin in NKCC1function, we studied a signaling mechanism linking NKCC1 and PKC.Stabilization of actin polymerization with jasplakinolide increasedactivity of NKCC1, whereas inhibition of actin polymerization withlatrunculin B prevented hormonal activation of NKCC1. Protein-proteininteractions among NKCC1, actin, and PKC- were verified by Westernblot analysis of immunoprecipitated proteins. PKC- was detected inimmunoprecipitates of NKCC1 and vice versa. Actin was also detected inimmunoprecipitates of NKCC1 and PKC-. Pulldown of endogenous actinrevealed the presence of NKCC1 and PKC-. Binding of recombinantPKC- to NKCC1 was not detected in overlay assays. Rather, activatedPKC- bound to actin, and this interaction was prevented by a peptideencoding C2, a C2-like domain based on the amino acid sequence ofPKC-. C2 also blocked stimulation of NKCC1 function bymethoxamine. Immunofluorescence and confocal microscopy revealedPKC- in the cytosol and cell periphery. Merged images of cellsstained for actin and PKC- indicated colocalization of PKC- andactin at the cell periphery. The results indicate that actin iscritical for the activation of NKCC1 through a direct interaction with PKC-.

     

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.

     

Inhibition of cell differentiation by Galpha q in the renal epithelial cell line LLC-PK1

LLC-PK₁, an epithelial cellline derived from the kidney proximal tubule, was used to study theability of the G protein -subunit, G_q, to regulate celldifferentiation. A constitutively active mutant protein,_qQ209L, was expressed using theLacSwitch-inducible mammalian expression system. Induction of_qQ209L expression with isopropyl--D-thiogalactopyranoside(IPTG) enhanced phospholipase C activity maximally by 6- to 7.5-fold.Increasing concentrations of IPTG progressively inhibited the activityof two differentiation markers,Na⁺-dependent hexose transport andalkaline phosphatase activity. Induction of_qQ209L expression also caused achange from an epithelial to a spindle-shaped morphology. The effectsof _qQ209L expression on celldifferentiation were similar to those observed with12-O-tetradecanoylphorbol 13-acetate(TPA) treatment. However, protein kinase C (PKC) levels weredownregulated in TPA-treated cells but not in_qQ209L-expressing cells,suggesting that the regulation of PKC byG_q may be different fromregulation by TPA. Interestingly, the PKC inhibitor GF-109203X did notinhibit the effect of IPTG on the development ofNa⁺-dependent hexose transport in_qQ209L-expressing cells. These data implicate PKC and PKC in the pathway used byG_q to block the development ofNa⁺-dependent hexose transport inIPTG-treated cells.

     

Osmotic pressure regulates alpha beta gamma -rENaC expressed in Xenopus oocytes

The hypothesisthat amiloride-sensitive Na⁺channels (ENaC) are involved in cell volume regulation was tested.Anisosmotic ND-20 media (ranging from 70 to 450 mosM) were used tosuperfuse Xenopus oocytes expressing-rat ENaC (-rENaC). Whole cell currents werereversibly dependent on external osmolarity. Under conditions ofswelling (70 mosM) or shrinkage (450 mosM), current amplitude decreasedand increased, respectively. In contrast, there was no change incurrent amplitude of H₂O-injectedoocytes to the above osmotic insults. Currents recorded from-rENaC-injected oocytes were not sensitive to externalCl concentration or to theK⁺ channel inhibitorBaCl₂. They were sensitive toamiloride. The concentration of amiloride necessary to inhibit one-halfof the maximal rENaC current expressed in oocytes(K_i; apparentdissociation constant) decreased in swollen cells and increased inshrunken oocytes. The osmotic pressure-inducedNa⁺ currents showed propertiessimilar to those of stretch-activated channels, including inhibition byGd³⁺ andLa³⁺, and decreased selectivityfor Na⁺.-rENaC-expressing oocytes maintained a nearly constant cell volume in hypertonic ND-20. The present study is the firstdemonstration that -rENaC heterologously expressed inXenopus oocytes may contribute tooocyte volume regulation following shrinkage.

     

A second enzyme protecting mineralocorticoid receptors from glucocorticoid occupancy

We have confirmed that A6 cells (derived fromkidney of Xenopus laevis), whichcontain both mineralocorticoid and glucocorticoid receptors, do notnormally possess 11-hydroxysteroid dehydroxgenase (11-HSD1 or11-HSD2) enzymatic activity and so are without apparent "protective" enzymes. A6 cells do not convert the glucocorticoid corticosterone to 11-dehydrocorticosterone but do, however, possess steroid 6-hydroxylase that transforms corticosterone to6-hydroxycorticosterone. This hydroxylase is cytochromeP-450 3A (CYP3A). We have nowdetermined the effects of 3,5-tetrahydroprogesterone andchenodeoxycholic acid (both inhibitors of 11-HSD1) and11-dehydrocorticosterone and11-hydroxy-3,5-tetrahydroprogesterone (inhibitors of11-HSD2) and carbenoxalone, which inhibits both 11-HSD1 and11-HSD2, on the actions and metabolism of corticosterone and activeNa⁺ transport [short-circuitcurrent(I_sc)] inA6 cells. All of these 11-HSD inhibitory substances induced asignificant increment in corticosterone-inducedI_sc, which wasdetectable within 2 h. However, none of these agents caused an increasein I_sc whenincubated by themselves with A6 cells. In all cases, the additionalI_sc was inhibitedby the mineralocorticoid receptor (MR) antagonist, RU-28318, whereasthe original I_scelicited by corticosterone alone was inhibited by the glucocorticoidreceptor antagonist, RU-38486. In separate experiments, each agent wasshown to significantly inhibit metabolism of corticosterone to6-hydroxycorticosterone in A6 cells, and a linear relationshipexisted between 6-hydroxylase inhibition and the MR-mediatedincrease in I_scin the one inhibitor tested. Troleandomycin, a selective inhibitor ofCYP3A, inhibited 6-hydroxylase and also significantly enhancedcorticosterone-induced I_sc at 2 h. Theseexperiments indicate that the enhanced MR-mediated I_sc in A6 cellsmay be related to inhibition of 6-hydroxylase activity in thesecells and that this 6-hydroxylase (CYP3A) may be protecting theexpression of corticosterone-induced active Na⁺ transport in A6 cells byMR-mediated mechanism(s).

     

Rat liver GTP-binding proteins mediate changes in mitochondrial membrane potential and organelle fusion

The variety of mitochondrial morphology in healthy and diseasedcells can be explained by regulated mitochondrial fusion. Previously, amitochondrial outer membrane fraction containing fusogenic, aluminumfluoride (AlF₄)-sensitiveGTP-binding proteins (mtg) was separated from rat liver (J. D. Cortese,Exp. Cell Res. 240: 122-133,1998). Quantitative confocal microscopy now reveals that mtgtransiently increases mitochondrial membrane potential () when added to permeabilized rat hepatocytes(15%), rat fibroblasts (19%), and rabbit myocytes (10%). This largemtg-induced increment is blocked by fusogenic GTPase-specificmodulators such as guanosine 5'-O-(3-thiotriphosphate),excess GTP (>100 µM), andAlF₄, suggesting a linkage between and mitochondrial fusion. Accordingly, stereometric analysisshows that decreasing or ATP synthesis with respiratory inhibitors limits mtg- andAlF₄-induced mitochondrial fusion. Also, a specific G protein inhibitor(Bordetellapertussis toxin) hyperpolarizesmitochondria and leads to a loss ofAlF₄-dependent mitochondrialfusion. These results place mtg-induced changes upstream ofAlF₄-induced mitochondrial fusion,suggesting that GTPases exert -dependent control of the fusionprocess. Mammalian mitochondrial morphology thus can be modulated bycellular energetics.     

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.     

Role of gelsolin in the actin filament regulation of cardiac L-type calcium channels

The actin cytoskeleton is an important contributor to themodulation of the cell function. However, little is known about theregulatory role of this supermolecular structure in the membrane eventsthat take place in the heart. In this report, the regulation of cardiacmyocyte function by actin filament organization was investigated inneonatal mouse cardiac myocytes (NMCM) from both wild-type mice andmice genetically devoid of the actin filament severing protein gelsolin(Gsn/). Cardiac L-type calcium channel currents(I_Ca) wereassessed using the whole cell voltage-clamp technique. Addition of theactin filament stabilizer phalloidin to wild-type NMCM increasedI_Ca by 227% overcontrol conditions. The basalI_Ca ofGsn/ NMCM was 300% higher than wild-type controls. Thisincrease was completely reversed by intracellular perfusion of theGsn/ NMCM with exogenous gelsolin. Further, cytoskeletal disruption of either Gsn/ or phalloidin-dialyzedwild-type NMCM with cytochalasin D (CD) decreased the enhancedI_Ca by 84% and 87%, respectively. The data indicate that actin filament stabilization by either a lack of gelsolin or intracellular dialysis with phalloidin increase I_Ca,whereas actin filament disruption with CD or dialysis ofGsn/ NMCM with gelsolin decreaseI_Ca. We concludethat cardiac L-type calcium channel regulation is tightly controlled byactin filament organization. Actin filament rearrangement mediated by gelsolin may contribute to calcium channel inactivation.

     

Regulation of a cloned epithelial Na+ channel by its beta - and gamma -subunits

Using the Xenopus oocyteexpression system, we examined the mechanisms by which the - and-subunits of an epithelial Na⁺channel (ENaC) regulate -subunit channel activity and the mechanisms by which -subunit truncations cause ENaC activation. Expression of-ENaC alone produced small amiloride-sensitive currents (43 ± 10 nA, n = 7). These currentsincreased >30-fold with the coexpression of - and -ENaC to1,476 ± 254 nA (n = 20).This increase was accompanied by a 3.1- and 2.7-fold increase ofmembrane fluorescence intensity in the animal and vegetal poles of theoocyte, respectively, with use of an antibody directed against the-subunit of ENaC. Truncation of the last 75 amino acids of the-subunit COOH terminus, as found in the original pedigree ofindividuals with Liddle's syndrome, caused a 4.4-fold(n = 17) increase of theamiloride-sensitive currents compared with wild-type -ENaC.This was accompanied by a 35% increase of animal pole membranefluorescence intensity. Injection of a 30-amino acid peptide withsequence identity to the COOH terminus of the human -ENaCsignificantly reduced the amiloride-sensitive currents by 40-50%.These observations suggest a tonic inhibitory role on the channel'sopen probability (P_o) by the COOH terminus of -ENaC. We conclude that the changes of current observed with coexpression of the - and -subunits or those observed with -subunit truncation are likely the result ofchanges of channel density in combination with large changes ofP_o.

     

CCK independently activates intracellular trypsinogen and NF-kappaB in rat pancreatic acinar cells

In the cholecystokinin (CCK)hyperstimulation model of acute pancreatitis, two early intracellularevents, activation of trypsinogen and activation of nuclear factor-B(NF-B), are thought to be important in the development of thedisease. In this study, the relationship between these two events wasinvestigated. NF-B activity was monitored by using a DNA bindingassay and mob-1 chemokine gene expression. Intracellulartrypsin activity was measured by using a fluorogenic substrate.Protease inhibitors including FUT-175, Pefabloc, and E-64d preventedCCK stimulation of intracellular trypsinogen and NF-B activation.Likewise, the NF-B inhibitors pyrrolidine dithiocarbamate andN-acetyl-L-cysteine inhibited CCK stimulation ofNF-B and intracellular trypsinogen activation. These resultssuggested a possible codependency of these two events. However, CCKstimulated NF-B activation in Chinese hamster ovary-CCK_Acells, which do not express trypsinogen, indicating that trypsin is notnecessary for CCK activation of NF-B. Furthermore,adenovirus-mediated expression in acinar cells of active p65 subunitsto stimulate NF-B, or of inhibitory B- molecules to inhibitNF-B, did not affect either basal or CCK-mediated trypsinogenactivation. Thus trypsinogen and NF-B activation are independentevents stimulated by CCK.

     

Acute glucose-induced downregulation of PKC-betaII accelerates cultured VSMC proliferation

Accelerated vascular smooth muscle cell(VSMC) proliferation contributes to the formation of atheroscleroticlesions. To investigate protein kinase C (PKC)-II functions withregard to glucose-induced VSMC proliferation, human VSMC from aorta(AoSMC), a clonal VSMC line of rat aorta (A10), and A10 cellsoverexpressing PKC-I (I-A10) and PKC-II (II-A10) werestudied with the use of three techniques to evaluate glucose effects onaspects affecting proliferation. High glucose (25 mM) increased DNAsynthesis and accelerated cell proliferation compared with normalglucose (5.5 mM) in AoSMC and A10 cells, but not in I-A10 andII-A10 cells. The PKC-II specific inhibitor CGP-53353 inhibitedglucose-induced cell proliferation and DNA synthesis in AoSMC and A10cells. In flow cytometry analysis, high glucose increased thepercentage of A10 cells at 12 h after cell cycle initiation butdid not increase the percentage of I-A10 or II-A10 cells enteringS phase. PKC-II protein levels decreased before the peak of DNAsynthesis, and high glucose further decreased PKC-II mRNA andprotein levels in AoSMC and A10 cells. These results suggest that highglucose downregulates endogenous PKC-II, which then alters thenormal inhibitory role of PKC-II in cell cycle progression,resulting in the stimulation of VSMC proliferation through acceleration of the cell cycle.

     

Effect of TNF-alpha on SMIT mRNA levels and myo-inositol accumulation in cultured endothelial cells

Previously we have shown that hyperosmolarity increasesNa⁺-myo-inositolcotransporter (SMIT) activity and mRNA levels in cultured endothelialcells. Because hyperosmolarity and cytokines, such as tumor necrosisfactor- (TNF-), activate similar signal transduction pathways, weexamined the effect of TNF- on SMIT mRNA levels andmyo-inositol accumulation. In contrastto the effect of hyperosmolarity, TNF- caused a time- andconcentration-dependent decrease in SMIT mRNA levels andmyo-inositol accumulation. The effectof TNF- on myo-inositolaccumulation was found in large-vessel endothelial cells (derived fromthe aorta and pulmonary artery) and cerebral microvessel endothelialcells. In bovine aorta and bovine pulmonary artery endothelial cells,TNF- activated nuclear factor (NF)-B. TNF- also increasedceramide levels, and C₂-ceramidemimicked the effect of TNF- on SMIT mRNA levels andmyo-inositol accumulation in bovineaorta endothelial cells. Pyrrolidinedithiocarbamate, genistein, and7-amino-1-chloro-3-tosylamido-2-hepatanone, compounds that can inhibitNF-B activation, partially prevented the TNF--induced decrease inmyo-inositol accumulation. The effectof TNF- on myo-inositolaccumulation was also partially prevented by the protein kinase Cinhibitor calphostin C but not by staurosporine. These studiesdemonstrate that TNF- causes a decrease in SMIT mRNA levels andmyo-inositol accumulation in culturedendothelial cells, which may be related to the activation of NF-B.

     

Colonic H-K-ATPase alpha- and beta-subunits express ouabain-insensitive H-K-ATPase

Active K absorption in the rat distal colon is energizedby an apical H-K-ATPase, a member of the gene family of P-type ATPases. The H-K-ATPase -subunit (HKc) has been cloned and characterized (together with the -subunit of either Na-K-ATPase or gastric H-K-ATPase) in Xenopus oocytes as ouabain-sensitive⁸⁶Rb uptake. In contrast, HKc, when expressed in Sf9cells without a -subunit, yielded evidence of ouabain-insensitiveH-K-ATPase. Because a -subunit (HKc) has recently been clonedfrom rat colon, this present study was initiated to determine whetherH-K-ATPase and its sensitivity to ouabain are expressed when these twosubunits (HKc and HKc) are transfected into a mammalian cellexpression system. Transfection of HEK-293 cells with HKc and HKccDNAs resulted in the expression of HKc and HKc proteins andtheir delivery to plasma membranes. H-K-ATPase activity was identified in crude plasma membranes prepared from transfected cells and was1) saturable as a function of increasing K concentration with aK_m for K of 0.63 mM; 2) inhibited byorthovanadate; and 3) insensitive to both ouabain andSch-28080. In parallel transfection studies with HKc and Na-K-ATPase1 cDNAs and with HKc cDNA alone, there was expression ofouabain-insensitive H-K-ATPase activity that was 60% and 21% of thatin HKc/HKc cDNA transfected cells, respectively. Ouabain-insensitive ⁸⁶Rb uptake was also identified incells transfected with HKc and HKc cDNAs. These studies establishthat HKc cDNA with HKc cDNA express ouabain-insensitiveH-K-ATPase similar to that identified in rat distal colon.