Inhibition of protein kinase D by resveratrol.

Protein kinase D (PKD) is a member of the protein kinase C (PKC) superfamily with distinctive structural, enzymological and regulatory properties. Identification of the cellular function(s) of PKD has been hampered by the absence of a selective inhibitor. Recently, Stewart et al. showed that resveratrol inhibited PKD, but not various PKC isoforms, in vitro. Here we confirmed that the activity of PKD is indeed inhibited in vitro by resveratrol (IC(50) approximately 200 microM). Additionally, we assessed the inhibition by resveratrol of PKD activity in intact cells, by Western blotting with a phosphospecific PKD antibody which recognizes the autophosphorylated enzyme. In this setting, very high concentrations of resveratrol were required to achieve inhibition of PKD autophosphorylation (IC(50) approximately 800 microM). Since resveratrol produces other pharmacological effects (e.g., cyclooxygenase inhibition) at lower concentrations than those required to inhibit PKD in intact cells, its value as a selective tool to investigate the cellular function(s) of PKD is questionable.

Expression and activity of protein kinase D/protein kinase C mu in myocardium: evidence for alpha1-adrenergic receptor- and protein kinase C-mediated regulation.

Protein kinase D (PKD), which is also known as protein kinase C (PKC) mu, is a novel serine/threonine kinase that can be activated in parallel with or downstream of PKC in various cell types, but its expression and regulation in myocardium have not been characterized. In the present study, two proteins of 110 and 115 kDa were detected in rat ventricular myocardium using antibodies directed at the extreme N- or C-terminus of PKD. Both proteins were highly expressed in the fetal heart but showed a developmental decline in abundance. Fractionation studies showed that PKD was distributed between myocyte and non-myocyte fractions in the neonatal heart, but was found predominantly in the non-myocyte fraction in the adult heart. In cultured neonatal rat ventricular myocytes, an in vitro kinase assay revealed increased autophosphorylation of PKD (EC50 2.8 nM) in response to phorbol-12-myristate-13-acetate (PMA). Exposure to norepinephrine also induced a dose-dependent increase in PKD autophosphorylation (EC50 0.6 microM). Pretreatment with the alpha1-adrenergic receptor (AR) antagonist prazosin blocked norepinephrine-induced PKD autophosphorylation, while the beta1-AR antagonist atenolol had no effect, indicating that activation of PKD by norepinephrine occurred via the alpha1-AR. Involvement of the alpha1-AR was confirmed by exposure of myocytes to the alpha1-AR agonist phenylephrine, which induced a similar profile of PKD autophosphorylation to norepinephrine (EC50 0.6 microM). The effects of both alpha1-AR stimulation and PMA on PKD autophosphorylation were mediated by PKC, since these effects could be attenuated by pretreatment of myocytes with the PKC inhibitor bisindolylmaleimide. These data show that PKD is expressed in rat ventricular myocardium, where its expression is subject to developmental control, and that PKD activity in ventricular myocytes is regulated through alpha1-AR- and PKC-mediated pathways.

Protein kinase D inhibits plasma membrane Na(+)/H(+) exchanger activity.

The regulation of plasma membrane Na(+)/H(+) exchanger (NHE) activity by protein kinase D (PKD), a novel protein kinase C- and phorbol ester-regulated kinase, was investigated. To determine the effect of PKD on NHE activity in vivo, intracellular pH (pH(i)) measurements were made in COS-7 cells by microepifluorescence using the pH indicator cSNARF-1. Cells were transfected with empty vector (control), wild-type PKD, or its kinase-deficient mutant PKD-K618M, together with green fluorescent protein (GFP). NHE activity, as reflected by the rate of acid efflux (J(H)), was determined in single GFP-positive cells following intracellular acidification. Overexpression of wild-type PKD had no significant effect on J(H) (3. 48 +/- 0.25 vs. 3.78 +/- 0.24 mM/min in control at pH(i) 7.0). In contrast, overexpression of PKD-K618M increased J(H) (5.31 +/- 0.57 mM/min at pH(i) 7.0; P < 0.05 vs. control). Transfection with these constructs produced similar effects also in A-10 cells, indicating that native PKD may have an inhibitory effect on NHE in both cell types, which is relieved by a dominant-negative action of PKD-K618M. Exposure of COS-7 cells to phorbol ester significantly increased J(H) in control cells but failed to do so in cells overexpressing either wild-type PKD (due to inhibition by the overexpressed PKD) or PKD-K618M (because basal J(H) was already near maximal). A fusion protein containing the cytosolic regulatory domain (amino acids 637-815) of NHE1 (the ubiquitous NHE isoform) was phosphorylated in vitro by wild-type PKD, but with low stoichiometry. These data suggest that PKD inhibits NHE activity, probably through an indirect mechanism, and represents a novel pathway in the regulation of the exchanger.

Regulation of sarcolemmal Na(+)/H(+) exchanger activity by angiotensin II in adult rat ventricular myocytes: opposing actions via AT(1) versus AT(2) receptors.

Increased sarcolemmal Na(+)/H(+) exchanger activity has been implicated as a mediator of the cardiac actions of angiotensin II. We studied the receptor subtypes and signaling pathways involved in the regulation of sarcolemmal Na(+)/H(+) exchanger activity by angiotensin II in adult rat ventricular myocytes. Cells were loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1, and acid efflux rates estimated during recovery from intracellular acidosis were used to quantify exchanger activity. Sarcolemmal Na(+)/H(+) exchanger activity was not affected by angiotensin II alone but was increased by angiotensin II plus PD123319 (AT(2) antagonist). In contrast, angiotensin II plus losartan (AT(1) antagonist) or CGP42112A (AT(2) agonist) did not affect exchanger activity. The increase in Na(+)/H(+) exchanger activity induced by angiotensin II plus PD123319 was blocked by losartan, PD98059 (extracellular signal-regulated kinase inhibitor), GF109203X (protein kinase C inhibitor), and tyrphostin AG1478 (epidermal growth factor receptor kinase inhibitor). Extracellular signal-regulated kinase phosphorylation and activity, measured by immunoblot analysis and an immune-complex kinase assay, respectively, were increased significantly by angiotensin II plus PD123319; these increases were blocked by losartan and PD98059. The increase in extracellular signal-regulated kinase phosphorylation induced by angiotensin II plus PD123319 was blocked also by GF109203X and tyrphostin AG1478. These data show that AT(1) stimulation increases sarcolemmal Na(+)/H(+) exchanger activity in adult rat ventricular myocytes and that this response requires extracellular signal-regulated kinase activation through a protein kinase C- and epidermal growth factor receptor-mediated mechanism. The positive effect of AT(1) stimulation on Na(+)/H(+) exchanger activity is counteracted by simultaneous AT(2) stimulation through a mechanism that does not involve direct inhibition of the exchanger or attenuation of extracellular signal-regulated kinase activation.

Regulation of cardiac sarcolemmal Na+/H+ exchanger activity by endogenous ligands. Relevance to ischemia.

The cardiac sarcolemmal Na+/H+ exchanger (NHE) extrudes one H+ in exchange for one Na+ entering the myocyte, utilizing for its driving force the inwardly directed Na+ gradient that is maintained by the Na+/K+ ATPase. The exchanger is quiescent at physiological values of intracellular pH but becomes activated in response to intracellular acidosis. Recent evidence suggests that a variety of extracellular signals (e.g., adrenergic agonists, thrombin, and endothelin) also modulate sarcolemmal NHE activity by altering its sensitivity to intracellular H+. Since sarcolemmal NHE activity is believed to be an important determinant of the extent of myocardial injury during ischemia and reperfusion, regulation of exchanger activity by endogenous ligands associated with ischemia is likely to be of pathophysiological importance.

Differential changes in transforming growth factor-beta isoform expression during postnatal cardiac growth.

Transforming growth factor-beta (TGF-beta) is synthesised as an inactive precursor protein; this is cleaved to produce the mature peptide and a latency associated protein (LAP), which remains associated with the mature peptide until activation by LAP degradation. Isoform specific antibodies raised against the LAPs for TGF-beta 2 and -beta 3 were used to determine the myocardial levels of LAP (activatable TGF-beta) and full length precursor (inactive TGF-beta) forms during post-natal development in the rat. TGF-beta 2 was present predominantly as the precursor in 2 day old myocardium. There was an age-dependent shift from precursor protein to LAP between 2 and 28 days. A corresponding increase in the level of mature (activatable) TGF-beta 2 was found. TGF-beta 3 was detected in significant quantities only as LAP. However, a four-fold increase in the expression of TGF-beta 3 LAP was observed between 2 and 28 days. The substantial increases in activatable forms of TGF-beta 2 and -beta 3 that occur in myocardium during the first 28 days of life in the rat support a role for these proteins in post-natal cardiac development.

Arterial disease and thrombosis in the antiphospholipid syndrome: a pathogenic role for endothelin 1.

OBJECTIVE: To explore a possible correlation between endothelin 1 (ET-1), the most potent endothelium-derived contracting factor that modulates vascular smooth muscle tone, and arterial disease in patients with the antiphospholipid syndrome (APS). METHODS: Plasma levels of ET-1 were measured in APS patients with (n = 16) and without (n = 11) arterial thrombosis and in non-APS patients with arterial thrombosis (n = 9). In addition, steady-state prepro-ET-1 messenger RNA (mRNA) levels were determined in endothelial cells treated with a range of human monoclonal anticardiolipin antibodies (aCL) (as anti-beta2-glycoprotein I antibodies) by semiquantitative 32P-dCTP-labeled reverse transcription-polymerase chain reaction. RESULTS: Compared with healthy controls, markedly increased plasma levels of ET-1 were found in APS patients with arterial thrombosis (2.00 +/- 0.87 versus 0.96 +/- 0.37 pg/ml; P = 0.0001) but not in other groups. Three human monoclonal aCL induced prepro-ET-1 mRNA levels significantly more than did control monoclonal antibody lacking aCL activity. CONCLUSION: Plasma ET-1 levels correlated significantly with a history of arterial thrombosis in patients with APS. Prepro-ET-1 mRNA was induced by human monoclonal aCL in the in vitro experimental system. The induction of ET-1 by antiphospholipid antibodies might contribute to increased arterial tone, leading to vasospasm and, ultimately, to arterial occlusion.

Cardiac Na+-H+ exchanger during postnatal development in the rat: changes in mRNA expression and sarcolemmal activity.

We examined Na+-H+ exchanger isoform 1 (NHE-1) mRNA expression in ventricular myocardium and its correlation with sarcolemmal NHE activity in isolated ventricular myocytes, during postnatal development in the rat. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA did not change in ventricular myocardium between 2 and 42 days after birth. Therefore, at seven time points within that age range. GAPDH expression was used to normalize NHE-1 mRNA levels, as determined by reverse transcription polymerase chain reaction analysis. There was a progressive five-fold reduction in NHE-1 mRNA expression in ventricular myocardium from 2 days to 42 days of age. As an index of NHE activity, acid efflux rates (J(H)) were determined in single neonatal (2-4-day-old) and adult (42-day-old) ventricular myocytes (n=16/group) loaded with the pH fluoroprobe carboxy-seminaphthorhodafluor-1. In HEPES-buffered medium, basal intracellular pH (pH(i)) was similar at 7.28+/-0.02 in neonatal and 7.31+/-0.02 in adult myocytes, but intrinsic buffering power was lower in the former age group. The rate at which pH(i) recovered from a similar acid load was significantly greater in neonatal than in adult myocytes (0.36+/-0.07 v 0.16+/-0.02 pH units/min at pH(i)=6.8). This was reflected by a significantly greater J(H) (22+/-4 v 9+/-1 pmol/cm2/s at pH(i)=6.8), indicating greater sarcolemmal NHE activity in neonatal myocytes. The concomitant reductions in tissue NHE-1 mRNA expression and sarcolemmal NHE activity suggest that myocardial NHE-1 is subject to regulation at the mRNA level during postnatal development.

Thrombin activates the sarcolemmal Na(+)-H+ exchanger. Evidence for a receptor-mediated mechanism involving protein kinase C.

Thrombin can activate the plasma membrane Na(+)-H+ exchanger in a variety of noncardiac cells. We have studied (1) the effect of thrombin on the activity of the sarcolemmal Na(+)-H+ exchanger in freshly isolated quiescent ventricular myocytes from the adult rat heart and (2) the signaling mechanism(s) underlying any effect. Reverse-transcription polymerase chain reaction analysis revealed thrombin receptor mRNA expression in a myocyte-enriched cell preparation. As an index of Na(+)-H+ exchanger activity, acid efflux rates (JHS) were determined in single myocytes (n = 4 to 11 per group) loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1 after two consecutive intracellular acid pulses (induced by transient exposure to 20 mmol/L NH4Cl) in bicarbonate-free medium. At a pHi of 6.9, JH did not change significantly during the second pulse relative to the first in control cells. However, when the second pulse occurred in the presence of 0.2, 1, or 5 U/mL thrombin, JH increased by 30%, 62% (P < .05), and 87% (P < .05), respectively. A hexameric thrombin receptor-activating peptide (SFLLRN) mimicked the effect of thrombin and increased JH by 73% (P < .05) at 25 mumol/L. In contrast, an inactive control peptide (FLLRN) was without effect at 25 mumol/L. In cells pretreated with 100 nmol/L GF109203X or 5 mumol/L chelerythrine (protein kinase C inhibitors), neither 5 U/mL thrombin nor 25 mumol/L SFLLRN produced a significant increase in JH. In the presence of 10 mumol/L HOE-694 (a Na(+)-H+ exchanger inhibitor), pHi did not recover after an acid load, even during exposure to 5 U/mL thrombin or 25 mumol/L SFLLRN, confirming that the Na(+)-H+ exchanger was the primary acid efflux mechanism under the conditions used. Neither 5 U/mL thrombin nor 25 mumol/L SFLLRN affected resting pHi and Ca2+ or background acid loading. We conclude that (1) adult rat ventricular myocytes express a functional thrombin receptor, whose stimulation results in increased activity of the sarcolemmal Na(+)-H+ exchanger, and (2) this effect appears to occur through a protein kinase C-mediated mechanism.

The carboxyl-terminal region of the Na+/H+ exchanger interacts with mammalian heat shock protein.

We expressed the carboxyl-terminal 178 amino acids of the rabbit cardiac Na+/H+ exchanger as a fusion protein with glutathione-S-transferase. The fusion protein (PCR178) was found in the supernatant of extracts of E. coli and was purified using Glutathione-Sepharose affinity chromatography. Affinity-purified antibodies raised against the carboxyl-terminal region of the Na+/H+ exchanger identified the resultant protein. PCR178 copurified with a 70 kDa protein. Amino-terminal sequencing of the 70 kDa protein identified it as dnaK, the bacterial equivalent of the mammalian 70 kDa heat shock protein (hsp70). DnaK was dissociated from the Na+/H+ exchanger fusion protein by the addition of MgATP. When purified PCR178 was coupled to a cyanogen bromide-activated Sepharose column, bovine hsp70 bound to the column and was eluted with MgATP. Nondenaturing polyacrylamide gel electrophoresis showed that, in the absence of MgATP, hsp70 formed a complex with PCR178. The complex was dissociated by the addition of MgATP. GST alone did not form a complex with hsp70. Immunoprecipitation of the Na+/H+ exchanger with antiexchanger antibodies resulted in coprecipitation of hsp70 protein from antiporter containing cells. Cells that overexpress the Na+/H+ exchanger had increased amounts of hsp70 which coprecipitated with antiexchanger antibody. The results show that heat shock protein complexes with the mammalian Na+/H+ exchanger.

Cloning and analysis of the human myocardial Na+/H+ exchanger.

The Na+/H+ exchanger is an integral membrane protein that is universally distribute in mammalian tissues and is responsible for intracellular pH regulation. Several isoforms of the Na+/H+ exchanger exist (NHE-1-NHE-4). The first that was cloned is the amiloride sensitive isoform (NHE-1). Using a fragment of the rabbit cardiac Na+/H+ exchanger cDNA clone we isolated and sequenced Na+/H+ exchanger cDNA from a human heart coding for the complete human Na+/H+ exchanger (NHE-1 isoform). Two overlapping cDNA clones were obtained, giving a combined sequence that contained both 3' and 5' untranslated regions. The 5' and 3' untranslated regions proved to be highly homologous to human sequences described earlier but contained some variations that could affect the mRNA stability and/or the efficiency of translation of the Na+/H+ exchanger. Northern blot analysis and reverse transcriptase polymerase chain reaction confirmed the presence of the 5 kb NHE-1 message in primary cultures of isolated myocytes.

Intracellular pH in Schizosaccharomyces pombe--comparison with Saccharomyces cerevisiae.

We examined cytoplasmic pH regulation in Schizosaccharomyces pombe and Saccharomyces cerevisiae using pH-sensitive fluorescent dyes. Of several different fluorescent compounds tested, carboxy-seminaphthorhodafluor-1 (C.SNARF-1) was the most effective. Leakage of C.SNARF-1 from S. pombe was much slower than leakage from C. cerevisiae. Using the pH-dependent fluorescence of C.SNARF-1 we showed that at an external pH of 7, mean resting internal pH was 7.0 for S. pombe and 6.6 for S. cerevisiae. We found that internal pH in S. pombe was maintained over a much narrower range in response to changes in external pH, especially at acidic pH. The addition of external glucose caused an intracellular alkalinization in both species, although the effect was much greater in S. cerevisiae than in S. pombe. The plasma membrane H(+)-ATPase inhibitor diethylstilbestrol reduced both the rate and extent of alkalinisation, with an IC50 of approximately 35 microM in both species. Amiloride also inhibited internal alkalinisation with IC50's of 745 microM for S. cerevisiae and 490 microM for S. pombe.

Multiple carbohydrate moieties on the Na+/H+ exchanger.

Affinity-purified antibodies against the C-terminal region of the Na+/H+ exchanger (NHE-1) were used to analyse the carbohydrate moiety of the protein. The Na+/H+ exchanger in human placental brush-border membranes has an apparent molecular mass of 105 kDa. Incubation of intact or detergent-solubilized membranes with glycopeptidase F removed the carbohydrate moiety and increased the apparent mobility of the exchanger. Digestion with endoglycosidase-F caused a similar change in mobility, but endoglycosidase-H had no effect, suggesting that the placental Na+/H+ exchanger is a glycoprotein of the biantennary complex type. Removal of the carbohydrate moiety with glycopeptidase F had no effect on the ability of the protein to promote the exchange of Na+ for H+, and had no detectable effect on the sensitivity of the exchanger to trypsin. Limited digestion with glycopeptidase F and neuraminidase indicated the presence of two intermediate forms between the fully glycosylated and the deglycosylated protein. This suggests the presence of at least two, and possibly three, N-linked carbohydrate moieties.

Amiloride and 5-(N-ethyl-N-isopropyl) amiloride inhibit medium acidification and glucose metabolism by the fission yeast Schizosaccharomyces pombe.

We have investigated the mechanism by which amiloride and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) inhibit glucose-stimulated medium acidification in the fission yeast Schizosaccharomyces pombe. The addition of glucose to an unbuffered suspension of cells results in the extrusion of acid. This process was inhibited by diethylstilbestrol (DES), an inhibitor of the H(+)-ATPase (IC50 71 microM), and also by amiloride (IC50 824 microM) and EIPA (IC50 203 microM). The presence of 100 mM NaCl reduced the degree of inhibition observed for amiloride and EIPA, but had no effect on inhibition by DES. N-Methylglucosamine partially protected the cells against the effect of amiloride, but choline chloride did not, suggesting that sodium may be important in the action of amiloride. To establish the site of action of amiloride and EIPA, ATP hydrolysis assays were performed on isolated plasma membranes. H(+)-ATPase activity was inhibited by orthovanadate, but not by amiloride or EIPA. However, both amiloride and EIPA were found to inhibit the incorporation of radioactivity from labelled glucose in S. pombe, with IC50 values of 879 and 272 microM for amiloride and EIPA respectively. Again, 100 mM NaCl was found to reduce the effectiveness of inhibition. Amiloride had no effect on the uptake of 2-deoxyglucose under the same conditions, indicating that amiloride does not inhibit the glucose transporter. We propose that amiloride and EIPA disrupt glucose-induced acidification by inhibiting glucose metabolism.

Characterization of the placental brush border membrane Na+/H+ exchanger: identification of thiol-dependent transitions in apparent molecular size.

We examined the protein and mRNA encoding the amiloride-sensitive Na+/H+ exchanger from human placenta. Reverse transcriptase PCR of human placental RNA and a human choriocarcinoma cell line showed that the message for the amiloride-sensitive Na+/H+ exchanger from human placenta. Reverse transcriptase PCR of human placental RNA and a human choriocarcinoma cell line showed that the message for the amiloride-sensitive Na+/H+ exchanger is present in the placenta and its derived cell line. Northern blot analysis showed only one species of Na+/H+ exchanger mRNA, of about 5 kb in size. To examine the Na+/H+ exchanger protein two different affinity-purified antibodies were produced against the C-terminal cytoplasmic region of the Na+/H+ exchanger. The antibodies both identified a 105 kDa protein in human placental brush border membrane vesicles. Under non-reducing conditions the amount of 105 kDa protein was greatly decreased, while a 205 kDa protein became apparent. This is probably a dimer of the 105 kDa protein. The monomer-to-dimer transition was dependent on the concentration of beta-mercaptoethanol. The results show that the amiloride-sensitive Na+/H+ exchanger is relatively abundant in human placenta and that it can exist as a larger 205 kDa protein linked by disulphide bonds.

Characterisation of proton fluxes across the cytoplasmic membrane of the yeast Saccharomyces cerevisiae.

We have tested the efficacy of fluorescent probes for the measurement of intracellular pH in Saccharomyces cerevisiae. Of the compounds tested (fluorescein, carboxyseminaphthorhodafluor-1 (C.SNARF-1) and 2',7'bis(carboxyethyl)-5(6')-carboxyfluorescein), C.SNARF-1 was found to be the most useful indicator of internal pH. Fluorescence microscopy showed that in Saccharomyces cerevisiae strain DAUL1, C.SNARF-1 and fluorescein had a heterogeneous distribution, with dye throughout the cytoplasm and concentration of the dye to an area close to the cell membrane. This region was also labeled by quinacrine, which is known to accumulate in acidic regions of the cell. Saccharomyces cerevisiae BJ4932, which carries a defect in vacuolar acidification, did not show the same degree of dye concentration, suggesting that the site of C.SNARF-1 and fluorescein localisation in DAUL1 is the acidic vacuole. Changes in intracellular pH could be monitored by measuring changes in the fluorescence intensity of C.SNARF-1. The addition of glucose caused an initial, rapid decrease in fluorescence intensity, indicating a rise in cellular pH. This was followed by slow acidification. Fluorescence intensity changes were similar in all strains studied, suggesting that the localisation of dye to acidic regions does not affect the measurement of intracellular pH in DAUL1. The changes in intracellular pH on the addition of glucose correlated well with glucose-induced changes in external pH. Preincubation of cells in the presence of the plasma membrane H(+)-ATPase inhibitor diethylstilbestrol reduced extracellular acidification and intracellular alkalinisation on the addition of glucose. Both amiloride and 5-(N-ethyl-N-isopropyl)amiloride also inhibited glucose-induced proton fluxes. Phorbol 12-myristate 13-acetate had no effect on the activity of the plasma membrane ATPase.

Uncoupler resistance in E. coli Tuv and Cuv is due to the exclusion of uncoupler by the outer membrane.

The uncoupler resistant bacterial strains E. coli Tuv and Cuv share the high deoxycholate sensitivity of the parent strain, Doc S. However, both Tuv and Cuv show greater resistance than Doc S to other detergents. Measurement of the periplasmic volume indicates that the outer membrane of Doc S is freely permeable to both TPP+ and hydroxymethylinulin. Tuv and Cuv are able to exclude these compounds. EDTA treatment was necessary prior to measuring membrane potential in Tuv and Cuv. Under conditions where delta phi could be measured, uncouplers acted to dissipate delta phi with equal potency in all strains. Uncoupler resistant proline uptake in Tuv and Cuv was abolished by EDTA treatment. Transduction experiments with phage P1 showed that uncoupler resistance could be transferred from Tuv to Doc S. Such transductants were no longer sensitive to novabiocin. The gene for uncoupler resistance cotransduced with the gene pyrE (82 min). Plating efficiency experiments with P1 suggests that detergent sensitivity in Doc S arises from an rfa (81 min) mutation. This mutation is no longer present in Tuv.

Uncoupler resistance in Escherichia coli: the role of cellular respiration.

Bioenergetic properties of a mutant strain of Escherichia coli K12 designated TUV, which is resistant to the protonophoric uncoupling agent 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidiazole (TTFB) have been compared with those of its non-resistant parent, E. coli K12 Doc-S. Strain TUV grew and respired some 20-30% faster than strain Doc-S, and was cross-resistant to carbonylcyanide p-(trifluoromethoxy)phenylhydrazone and triphenyltin, but not to 2,4-dinitrophenol. Phosphorus nuclear magnetic resonance demonstrated the TTFB-mediated collapse of the transmembrane pH gradient at identical rates in starved cells of both strains, indicating that uncoupler access and function were unimpaired in the mutant under these conditions. Strain TUV displayed enhanced uncoupler resistance and maintained intracellular pH and ATP levels only when respiring. On the other hand, strain TUV also showed increased resistance to novobiocin, implying that its outer wall permeability had been lowered. We suggest that the active resistance of strain TUV results from the exclusion of uncoupler by the interaction of inner and outer membrane components in a manner modulated by the degree of cellular energization.