Electrophysiologie des Membranes

RESUMES

Mol Plant Microbe Interact. 2006 May ;19:550-6

A putative role for fusaric acid in biocontrol of the parasitic angiosperm Orobanche ramosa.

Brahim Bouizgarne, Hayat El-Maarouf-Bouteau, Karine Madiona, Bernadette Biligui, Michèle Monestiez, Anne Marie Pennarun, Zahia Amiar, Jean Pierre Rona, Yedir Ouhdouch, Ismaïl El Hadrami, François Bouteau

Fusarium spp. are ubiquitous fungi found in soil worldwide as both pathogenic and nonpathogenic strains. The signals leading to disease or the absence of disease are poorly understood. We recently showed that fusaric acid (FA), a nonspecific toxin produced by most Fusarium spp., could elicit various plant defense responses at 100 nM without toxic effect. In this study, we checked for the effect of FA on root and root hairs, probable first site of contact between the fungi and the host. Large FA concentrations reduce root and root-hair growth and induce a rapid transient membrane hyperpolarization, followed by a large depolarization, due to the inhibition of H(+)-ATPase currents. Nanomolar concentrations of FA induced only an early transient membrane hyperpolarization of root hairs compatible with the induction of a signal transduction pathway. FA at 10(-7) M failed to induce salicylic acid- and jasmonic acid/ethylene-dependent defense-related genes but inhibited the germination of the angiosperm parasite Orobanche ramosa in contact of FA-pretreated Arabidopsis thaliana seedlings. These data suggest that FA at nontoxic concentrations could activate signal transduction components necessary for plant-defense responses that could contribute to biocontrol activity of Fusarium spp.

New Phytol. 2006 Jan ;169:209-18

Early physiological responses of Arabidopsis thaliana cells to fusaric acid: toxic and signalling effects.

B Bouizgarne, H El-Maarouf-Bouteau, C Frankart, D Reboutier, K Madiona, A M Pennarun, M Monestiez, J Trouverie, Z Amiar, J Briand, M Brault, J P Rona, Y Ouhdouch, I El Hadrami, F Bouteau

* Fusaric acid (FA) is a toxin produced by Fusarium species. Most studies on FA have reported toxic effects (for example, alteration of cell growth, mitochondrial activity and membrane permeability) at concentrations greater than 10(-5) m. FA participates in fungal pathogenicity by decreasing plant cell viability. However, FA is also produced by nonpathogenic Fusarii, potential biocontrol agents of vascular wilt fusaria. The aim of this study was to determine whether FA, at nontoxic concentrations, could induce plant defence responses. * Nontoxic concentrations of FA were determined from cell-growth and O(2)-uptake measurements on suspensions of Arabidopsis thaliana cells. Ion flux variations were analysed from electrophysiological and pH measurements. H(2)O(2) and cytosolic calcium were quantified by luminescence techniques. * FA at nontoxic concentrations (i.e. below 10(-6) m) was able to induce the synthesis of phytoalexin, a classic delayed plant response to pathogen. FA could also induce rapid responses putatively involved in signal transduction, such as the production of reactive oxygen species, and an increase in cytosolic calcium and ion channel current modulations. * FA can thus act as an elicitor at nanomolar concentrations.

Plant Cell Physiol. 2005 Sep ;46:1494-504

Brassinosteroids regulate plasma membrane anion channels in addition to proton pumps during expansion of Arabidopsis thaliana cells.

Zongshen Zhang, Javier Ramirez, David Reboutier, Mathias Brault, Jacques Trouverie, Anne-Marie Pennarun, Zahia Amiar, Bernadette Biligui, Lydia Galagovsky, Jean-Pierre Rona

Brassinosteroids (BRs) are involved in numerous physiological processes associated with plant development and especially with cell expansion. Here we report that two BRs, 28-homobrassinolide (HBL) and its direct precursor 28-homocastasterone (HCS), promote cell expansion of Arabidopsis thaliana suspension cells. We also show that cell expansions induced by HBL and HCS are correlated with the amplitude of the plasma membrane hyperpolarization they elicited. HBL, which promoted the larger cell expansion, also provoked the larger hyperpolarization. We observed that membrane hyperpolarization and cell expansion were partially inhibited by the proton pump inhibitor erythrosin B, suggesting that proton pumps were not the only ion transport system modulated by the two BRs. We used a voltage clamp approach in order to find the other ion transport systems involved in the PM hyperpolarization elicited by HBL and HCS. Interestingly, while anion currents were inhibited by both HBL and HCS, outward rectifying K+ currents were increased by HBL but inhibited by HCS. The different electrophysiological behavior shown by HBL and HCS indicates that small changes in the BR skeleton might be responsible for changes in bioactivity.

Plant Physiol Biochem. 2005 Jun ;43:567-72

A CFTR chloride channel activator prevents HrpN ea-induced cell death in Arabidopsis thaliana suspension cells.

David Reboutier, Cécile Frankart, Régine Vedel, Mathias Brault, Ronald G Duggleby, Jean Pierre Rona, Marie Anne Barny, François Bouteau

Erwinia amylovora is a necrogenic bacterium that causes fire blight of the Maloideae subfamily of Roseacae, such as apple and pear. It provokes necrosis in aerial parts of susceptible host plants and the typical hypersensitive reaction in non-host plants. The secreted harpin, HrpN ea, is able by itself to induce an active cell death in non-host plants. Ion flux modulations were shown to be involved early in such processes but very few data are available on the plasma membrane ion channel activities responsible for the pathogen-induced ion fluxes. We show here that HrpN ea induces cell death in non-host Arabidopsis thaliana suspension cells. We further show that two cystic fibrosis transmembrane conductance regulator modulators, glibenclamide and bromotetramisole, can regulate anion channel activities and HrpN ea-induced cell death.

Plant J. 2005 Apr ;42:145-52

Diacylglycerol pyrophosphate is a second messenger of abscisic acid signaling in Arabidopsis thaliana suspension cells.

Christine Zalejski, Zongshen Zhang, Anne-Laure Quettier, Régis Maldiney, Magda Bonnet, Mathias Brault, Chantal Demandre, Emile Miginiac, Jean-Pierre Rona, Bruno Sotta, Emmanuelle Jeannette

In plants, the importance of phospholipid signaling in responses to environmental stresses is becoming well documented. The involvement of phospholipids in abscisic acid (ABA) responses is also established. In a previous study, we demonstrated that the stimulation of phospholipase D (PLD) activity and plasma membrane anion currents by ABA were both required for RAB18 expression in Arabidopsis thaliana suspension cells. In this study, we show that the total lipids extracted from ABA-treated cells mimic ABA in activating plasmalemma anion currents and induction of RAB18 expression. Moreover, ABA evokes within 5 min a transient 1.7-fold increase in phosphatidic acid (PA) followed by a sevenfold increase in diacylglycerol pyrophosphate (DGPP) at 20 min. PA activated plasmalemma anion currents but was incapable of triggering RAB18 expression. By contrast, DGPP mimicked ABA on anion currents and was also able to stimulate RAB18 expression. Here we show the role of DGPP as phospholipid second messenger in ABA signaling.

Plant Physiol. 2004 May ;135:231-43

Plasma membrane depolarization induced by abscisic acid in Arabidopsis suspension cells involves reduction of proton pumping in addition to anion channel activation, which are both Ca2+ dependent.

Mathias Brault, Zahia Amiar, Anne-Marie Pennarun, Michèle Monestiez, Zongshen Zhang, Daniel Cornel, Olivier Dellis, Heather Knight, François Bouteau, Jean-Pierre Rona

In Arabidopsis suspension cells a rapid plasma membrane depolarization is triggered by abscisic acid (ABA). Activation of anion channels was shown to be a component leading to this ABA-induced plasma membrane depolarization. Using experiments employing combined voltage clamping, continuous measurement of extracellular pH, we examined whether plasma membrane H(+)-ATPases could also be involved in the depolarization. We found that ABA causes simultaneously cell depolarization and medium alkalinization, the second effect being abolished when ABA is added in the presence of H+ pump inhibitors. Inhibition of the proton pump by ABA is thus a second component leading to the plasma membrane depolarization. The ABA-induced depolarization is therefore the result of two different processes: activation of anion channels and inhibition of H(+)-ATPases. These two processes are independent because impairing one did not suppress the depolarization. Both processes are however dependent on the [Ca2+]cyt increase induced by ABA since increase in [Ca(2+)](cyt) enhanced anion channels and impaired H(+)-ATPases.

Plant Physiol. 2002 Sep ;130:265-72

Plasmalemma abscisic acid perception leads to RAB18 expression via phospholipase D activation in Arabidopsis suspension cells.

Matthieu Hallouin, Thanos Ghelis, Mathias Brault, Françoise Bardat, Daniel Cornel, Emile Miginiac, Jean-Pierre Rona, Bruno Sotta, Emmanuelle Jeannette

Abscisic acid (ABA) plays a key role in the control of stomatal aperture by regulating ion channel activities and water exchanges across the plasma membrane of guard cells. Changes in cytoplasmic calcium content and activation of anion and outward-rectifying K(+) channels are among the earliest cellular responses to ABA in guard cells. In Arabidopsis suspension cells, we have demonstrated that outer plasmalemma perception of ABA triggered similar early events. Furthermore, a Ca(2+) influx and the activation of anion channels are part of the ABA-signaling pathway leading to the specific expression of RAB18. Here, we determine whether phospholipases are involved in ABA-induced RAB18 expression. Phospholipase C is not implicated in this ABA pathway. Using a transphosphatidylation reaction, we show that ABA plasmalemma perception results in a transient stimulation of phospholipase D (PLD) activity, which is necessary for RAB18 expression. Further experiments showed that PLD activation was unlikely to be regulated by heterotrimeric G proteins. We also observed that ABA-dependent stimulation of PLD was necessary for the activation of plasma anion current. However, when ABA activation of plasma anion channels was inhibited, the ABA-dependent activation of PLD was unchanged. Thus, we conclude that in Arabidopsis suspension cells, ABA stimulation of PLD acts upstream from anion channels in the transduction pathway leading to RAB18 expression.

Mol Plant Microbe Interact. 2002 Sep ;15:932-8

The indolic compound hypaphorine produced by ectomycorrhizal fungus interferes with auxin action and evokes early responses in nonhost Arabidopsis thaliana.

David Reboutier, Michele Bianchi, Mathias Brault, Camille Roux, Aurélien Dauphin, Jean-Pierre Rona, Valérie Legué, Frédéric Lapeyrie, François Bouteau

Signals leading to mycorrhizal differentiation are largely unknown. We have studied the sensitivity of the root system from plant model Arabidopsis thaliana to hypaphorine, the major indolic compound isolated from the basidiomycetous fungus Pisolithus tinctorius. This fungi establishes ectomycorrhizas with Eucalyptus globulus. Hypaphorine controls root hair elongation and counteracts the activity of indole-3-acetic acid on root elongation on A. thaliana, as previously reported for the host plant. In addition, we show that hypaphorine counteracts the rapid upregulation by indole-3-acetic acid and 1-naphthalenic-acetic acid of the primary auxin-responsive gene IAA1 and induces a rapid, transient membrane depolarization in root hairs and suspension cells, due to the modulation of anion and K+ currents. These early responses indicate that components necessary for symbiosis-related differentiation events are present in the nonhost plant A. thaliana and provide tools for the dissection of the hypaphorine-auxin interaction.

J Biol Chem. 2002 Feb 22;277:6044-50

Inhibition of the calcium release-activated calcium (CRAC) current in Jurkat T cells by the HIV-1 envelope protein gp160.

Olivier Dellis, Sophie C Gangloff, Marc Paulais, Danielle Tondelier, Jean-Pierre Rona, Franck Brouillard, François Bouteau, Moncef Guenounou, Jacques Teulon

The HIV-1 envelope glycoprotein gp120/160 has pleiotropic effects on T cell function. We investigated whether Ca(2+) signaling, a crucial step for T cell activation, was altered by prolonged exposure of Jurkat T cells to gp160. Microfluorometric measurements showed that Jurkat cells incubated with gp160 had smaller (approximately 40%) increases in [Ca(2+)](i) in response to phytohemagglutinin and had a reduced Ca(2+) influx (approximately 25%). gp160 had similar effects on Jurkat cells challenged with thapsigargin. We used the patch clamp technique to record the Ca(2+) current, which is responsible for Ca(2+) influx and has properties of the calcium release-activated Ca(2+) current (I(CRAC)). gp160 reduced I(CRAC) by approximately 40%. The inhibitory effects of gp160 were antagonized by staurosporine (0.1 microm), an inhibitor of protein-tyrosine kinases and protein kinase Cs (PKCs), and by Gö 6976 (5 microm), an inhibitor acting especially on PKC alpha and PKC beta I. 12-O-Tetradecanoyl phorbol 13-acetate (16 nm), a PKC activator, reproduced the effects of gp160 in untreated cells. A Western blotting analysis of PKC isoforms alpha, beta I, delta, and zeta showed that only the cellular distribution of PKC alpha and -beta I were significantly modified by gp160. In addition, gp160 was able to modify the subcellular distribution of PKC alpha and PKC beta I caused by phytohemagglutinin. Therefore the reduction in I(CRAC) caused by prolonged incubation with gp160 is probably mediated by PKC alpha or -beta I.

FEBS Lett. 2001 May 25;497:82-4

Harpin, a hypersensitive response elicitor from Erwinia amylovora, regulates ion channel activities in Arabidopsis thaliana suspension cells.

H El-Maarouf, M A Barny, J P Rona, F Bouteau

HrpN, the hypersensitive response elicitor from Erwinia amylovora, stimulated K(+) outward rectifying currents in Arabidopsis thaliana suspension cells. It also decreased anion currents. These data demonstrate the ability of harpin to regulate different plasma membrane ion channels, putative components of signal transduction chains leading to defense responses and programmed cell death.

FEBS Lett. 2000 May 26;474:43-7

Abscissic acid specific expression of RAB18 involves activation of anion channels in Arabidopsis thaliana suspension cells.

T Ghelis, O Dellis, E Jeannette, F Bardat, D Cornel, E Miginiac, J Rona, B Sotta

The abscissic acid (ABA) transduction cascade following the plasmalemma perception was analyzed in intact Arabidopsis thaliana suspension cells. In response to impermeant ABA, anion currents were activated and K(+) inward rectifying currents were inhibited. Anion current activation was required for the ABA specific expression of RAB18. By contrast, specific inhibition of K(+) channels by tetraethylammonium or Ba(2+) did not affect RAB18 expression. Thus, outer plasmalemma ABA perception triggered two separated signaling pathways.

Hepatology. 2000 Nov ;32:935-41

Evidence for an endothelium-derived hyperpolarizing factor in the superior mesenteric artery from rats with cirrhosis.

E Barriere, K A Tazi, J P Rona, F Pessione, J Heller, D Lebrec, R Moreau

In cirrhosis, in splanchnic arteries, endothelium-dependent relaxation may persist even if overactive nitric oxide synthase (NOS) and cyclooxygenase (COX) are inhibited. In normal arteries, a significant endothelium-dependent relaxation to acetylcholine persists after NOS/COX inhibition. This relaxation is caused by smooth muscle cell (SMC) membrane hyperpolarization, which is sensitive to a combination of the potassium channel blockers apamin and charybdotoxin, and is mediated by an endothelium-derived hyperpolarizing factor (EDHF). The aim of this study was to detect EDHF and evaluate its pathophysiologic role in isolated superior mesenteric arteries from cirrhotic rats. Arterial rings were obtained and exposed to N(w)-nitro-L-arginine (L-NNA, a NOS inhibitor) and indomethacin (a COX inhibitor). Acetylcholine-induced membrane potential responses and concentration-response curves to the relaxant of acetylcholine were obtained with and without apamin plus charybdotoxin. Acetylcholine-induced responses were measured in certain rings from endothelium-denuded arteries. Contractions caused by the alpha(1)-adrenoceptor agonist phenylephrine were obtained in cirrhotic and normal rings with and without apamin and charybdotoxin. Significant acetylcholine-induced, endothelium-dependent, apamin- and charybdotoxin-sensitive, SMC membrane hyperpolarization and relaxation were found. An apamin- and charybdotoxin-sensitive hyporesponsiveness to the contractile action of phenylephrine was found in cirrhotic rings. In conclusion, in cirrhotic rats, in the superior mesenteric artery exposed to NOS/COX-inhibitors, an EDHF exists that may replace NOS/COX products to induce endothelium-dependent arterial relaxation.

Plant J. 2000 Apr ;22:9-17

Ion currents involved in early Nod factor response in Medicago sativa root hairs: a discontinuous single-electrode voltage-clamp study.

A Kurkdjian, F Bouteau, A M Pennarun, M Convert, D Cornel, J P Rona, U Bousquet

Nod factor [NodRm-IV(Ac,S)], isolated from the bacterium Rhizobium meliloti, induces a well-known depolarization in Medicago sativa (cv Sitel) root hairs. Analysis of this membrane response using the discontinuous single-electrode voltage-clamp technique (dSEVC) shows that anion channel, K+ channel and H+-ATPase pump currents are involved in young growing root hairs. The early Nod-factor-induced depolarization is due to increase of the inward ion current and inhibition of the H+ pump. It involved an instantaneous inward anion current (IIAC) and/or a time-dependent inward K+ current (IRKC). These two ion currents are then down-regulated while the H+ pump is stimulated, allowing long-term rectification of the membrane potential (Em). Our results support the idea that the regulation of inward current plays a primary role in the Nod-factor-induced electrical response, the nature of the ions carried by these currents depending on the activated anion and/or K+ channels at the plasma membrane.

FEBS Lett. 1999 Sep 17;458:185-7

Transient outward K+ currents across the plasma membrane of laticifer from Hevea brasiliensis.

F Bouteau, O Dellis, J P Rona

Non-inactivating outward rectifying K+ channel currents have been identified in a variety of plant cell types and species. The present study of laticifer protoplasts from Hevea brasiliensis, cells which are specialized for stress response, has revealed, through a switch-clamp method, an outward rectifying current displaying rapid inactivation. The inactivation depended on the external K+ concentration and on the voltage. This current inactivation appeared clearly different from all those previously described in plant cells and it shared homology with current kinetics of animal Shaker family channels. These results, given the recent cloning of plant K+ channel beta-subunits, shed new light on possible plant K+ channel regulation.

Plant J. 1999 Apr ;18:13-22

Induction of RAB18 gene expression and activation of K+ outward rectifying channels depend on an extracellular perception of ABA in Arabidopsis thaliana suspension cells.

E Jeannette, J P Rona, F Bardat, D Cornel, B Sotta, E Miginiac

Important progress has been made regarding the characterization of the ABA signalling components using genetic and molecular approaches (Leung and Giraudat, 1998). However, we do not yet know the mechanism of ABA perception. Conflicting results concerning the site of ABA perception have been published. The prevailing view is that since ABA controls many responses, different sites of perception for ABA might exist. In order to establish the cellular localisation of the ABA receptors in Arabidopsis thaliana suspension cells, we developed two physiological tests based upon the capacity of impermeant ABA-BSA conjugate to mimic permeant free ABA effects. We show that purified ABA-BSA conjugate is able to trigger RAB18 gene expression and that this response is strictly due to the natural (+)-ABA enantiomer. The rate of RAB18 gene expression was independent of the level of ABA uptake by the cells. Using the voltage-clamp technique we show that ABA-BSA, similarly to ABA, evokes a membrane depolarization and activates time- and voltage-dependent outward rectifying currents (ORC). We demonstrate that these ORC are due to a K+ efflux as assessed by tail currents and specific inhibition by both tetraethylammonium (TEA) and Ba2+. These observations provide evidence in favour of an extracellular site for ABA perception.

Bioelectrochem Bioenerg. 1999 Feb ;48:135-9

Evidence of multiple sugar uptake across the plasma membrane of laticifer protoplasts from Hevea.

F Bouteau, O Dellis, U Bousquet, J P Rona

In Hevea, rubber synthesis is confined to the cytosol of the highly differentiated laticifer cells. Agronomic and biochemical studies showed that this process uses high amounts of sugars that are efficiently imported into the laticifer. A H(+)-sugar symport system located in the plasma membrane is involved in sugar uptake into laticifers. Laticifer protoplasts were prepared and used in electrophysiological and labeling experiments to test the capacity of this system to transport a variety of sugars such as oligosaccharides from the raffinose family, trace compounds in rubber. Translocation of sugars known to be transported with different efficiency across the plasma membrane of plant cells was also tested. A 1 mM sucrose affinity was found for the symport. All the sugars tested, except palatinose induce membrane depolarization indicating that they were actively absorbed by the laticifer network. This reveals the wide capacity of this peculiar sink for the uptake of sugars.

FEBS Lett. 1999 Jan 25;443:187-91

HIV-1 gp160 decreases the K+ voltage-gated current from Jurkat E6.1 T cells by up-phosphorylation.

O Dellis, F Bouteau, M Guenounou, J P Rona

HIV-1 gp120/gp160 is known to disturb the activity of p56lck, protein kinase C (PKC) and Ca2+ homeostasis in T lymphocytes. We found that gp160 decreases the Kv1.3 current of Jurkat E6.1 cells probably by increasing the PKC-dependent phosphorylation of Kv channel protein after 5 days. This decrease is dose-dependent. In contrast, gp160 did not decrease the Kv1.3 current of the JCaM1.6 cell line, a p56(lck)-defective Jurkat cell line. This shows that p56lck was at the beginning of the events which induced the Kv1.3 current decrease. As a consequence of this decrease, Jurkat E6.1 cells were depolarized and exhibited a volume increase.

J Hepatol. 1999 Jan ;30:107-14

Endothelium-dependent blunted membrane potential responses to ATP-sensitive K+ channel modulators in aortae from rats with cirrhosis.

P Lahaye, L Fouassier, K A Tazi, A De Gottardi, J F Fléjou, C Chagneau, J P Rona, C Housset, J Reichen, D Lebrec, R Moreau

BACKGROUND/AIMS: In vivo studies have shown that arterial vasodilation induced by synthetic openers of ATP-sensitive K+ (K(ATP)) channels is decreased in rats with cirrhosis. Since vasodilation induced by these substances is mediated by membrane potential hyperpolarization in arterial smooth muscle cells, membrane potential hyperpolarization in response to K(ATP) channel openers may be altered in cirrhotic smooth muscle cells. The aim of the present study was to investigate the effects of K(ATP) channel modulators (i.e. openers and blockers of these channels) on the membrane potential in smooth muscle cells in isolated aortae from cirrhotic and normal rats. The influence of endothelin-1 production by endothelial cells on smooth muscle cells membrane potential responses to K(ATP) channel modulators was also studied. METHODS: Cells were impaled in situ (in intact and endothelium-denuded aortae) with a microelectrode that was used to measure membrane potentials. K(ATP) channel openers were diazoxide or cromakalim; blockers were glibenclamide or tolbutamide. Bosentan (a mixed endothelin receptor antagonist) and exogenous endothelin-1 were also used. Preproendothelin-1 mRNA was assayed in aortae by RNase protection assay. Aortic wall endothelin-1 concentration was measured by double antibody radioimmunoassay technique. RESULTS: As expected, in smooth muscle cells in intact normal aortae, K(ATP) channel openers induced membrane potential hyperpolarization and K(ATP) channel blockers membrane potential depolarization. In smooth muscle cells in intact cirrhotic aortae, K(ATP) channel openers and blockers did not significantly change the membrane potential. Endothelium removal or exposure of intact aortae to bosentan restored normal membrane potential responses to K(ATP) channel modulators in cirrhotic smooth muscle cells and did not alter the effects of these substances in normal smooth muscle cells. In endothelium-denuded aortae, exposure to exogenous endothelin-1 suppressed membrane potential responses to K(ATP) channel modulators. In intact aortae, the abundance of preproendothelin-1 mRNA and endothelin-1 did not significantly differ between normal and cirrhotic rats. CONCLUSIONS: K(ATP) channel opener-induced membrane hyperpolarization and K(ATP) channel blocker-elicited membrane depolarization are blunted in smooth muscle cells in intact cirrhotic aortae. This blunting is due to the activation of the endothelin-1 pathway in the aortic wall, downstream to the endothelial production of endothelin-1.

Hepatology. 1998 Sep ;28:663-9

Effects of protein kinase C modulators on Na+/K+ adenosine triphosphatase activity and phosphorylation in aortae from rats with cirrhosis.

P Lahaye, K A Tazi, J P Rona, O Dellis, D Lebrec, R Moreau

Protein kinase C (PKC) modulates the activity and phosphorylation of the catalytic alpha-subunit of sodium-potassium-adenosine triphosphatase (Na+/K+ ATPase) in normal arteries. Because PKC is altered in cirrhotic aortae, Na+/K+ ATPase may also be altered in these arteries. The aim of the present study was to investigate alpha-subunit activity and phosphorylation in aortae from normal and cirrhotic rats, under baseline conditions and during exposure to PKC modulators. Alpha-subunit activity was assessed by measuring the amount of 32P released by hydrolysis of [gamma-32P]ATP in freshly isolated cell membranes (in the absence of PKC modulators only) and membrane depolarization caused by ouabain-induced alpha-subunit inhibition in isolated aortae (in the absence and presence of PKC modulators). Alpha-subunit phosphorylation was assessed by incorporation of 32P into alpha-subunits. Staurosporine, a PKC inhibitor, and phorbol 12,13-dibutyrate (PDBU), a PKC activator, were used. In addition, alpha-subunit expression was studied by Western blot analysis. In the absence of PKC modulators, the amount of 32P released by hydrolysis of [gamma-32P]ATP and ouabain-induced membrane depolarization were significantly lower in cirrhotic than in normal aortae. Staurosporine suppressed ouabain-induced membrane depolarization in cirrhotic and normal arteries. Ouabain-induced membrane depolarization was similar in cirrhotic aortae exposed to PDBU and in normal arteries studied under baseline conditions. Alpha-subunit phosphorylation was significantly lower in cirrhotic than in normal aortae, in aortae under baseline conditions, and in arteries exposed to staurosporine. Phosphorylation of the alpha-subunit was similar in cirrhotic aortae exposed to PDBU and in normal arteries under baseline conditions. Western blot analysis showed that the amount of alpha-subunit did not significantly differ between cirrhotic and normal aortae. In conclusion, a decrease in baseline Na+/K+ ATPase alpha-subunit activity occurs in aortae from cirrhotic rats as a result of reduced basal PKC activity. This PKC-dependent decreased alpha-subunit activity may be caused by a reduction in PKC-induced alpha-subunit phosphorylation.