Evidence for ΔpH surface component (ΔpH) of proton motive force in ATP synthesis of mitochondria

Background

One of the central debates in membrane bioenergetics is whether proton-dependent energy coupling mechanisms are mediated exclusively by protonic transmembrane electrochemical potentials, as delocalized pmf, ΔµH⁺, or by more localized membrane surface proton pathways, as interfacial pmf, ΔµH^S.

Methods

We measure ?pH^S in rat liver mitoplasts energized by respiration or ATP hydrolysis by inserting pH sensitive fluorescein-phosphatidyl-ethanolamine(F-PE) into mitoplast surface.

Results

In the presence of rotenone and Ap5A, succinate oxidation induces a bi-phasic interfacial protonation on the mitoplast membranes, a fast phase followed by a slow one, and an interfacial pH decrease of 0.5 to 0.9 pH units of mitoplast with no simultaneous pH changes in the bulk. Antimycin A, other inhibitors or uncouplers of mitochondrial respiration prevent the decrease of mitoplast ?pH^S, supporting that ΔµH^S is dependent and controlled by energization of mitoplast membranes. A quantitative assay of ATP synthesis coupled with ?pH^S of mitoplasts oxidizing succinate with malonate titration shows a parallel correlation between ATP synthesis, State 4 respiration and ?pH^S, but not with ?Ψ_E.

General Significance

Our data substantiate ?pH^S as the primary energy source of pmf for mitochondrial ATP synthesis. Evidence and discussion concerning the relative importance and interplay of ?pH^S and ?Ψ_E in mitochondrial bioenergetics are also presented.     

Suppression of ρ 0 lethality by mitochondrial ATP synthase F1 mutations in Kluyveromyces lactis occurs in the absence of F0

Specific mgi mutations in the α, β or γ subunits of the mitochondrial F₁-ATPase have previously been found to suppress ρ⁰ lethality in the petite-negative yeast Kluyveromyces lactis. To determine whether the suppressive activity of the altered F₁ is dependent on the F₀ sector of ATP synthase, we isolated and disrupted the genes KlATP4, 5 and 7, the three nuclear genes encoding subunits b, OSCP and d. Strains disrupted for any one, or all three of these genes are respiration deficient and have reduced viability. However a strain devoid of the three nuclear genes is still unable to lose mitochondrial DNA, whereas a mgi mutant with the three genes inactivated remains petite-positive. In the latter case, ρ⁰ mutants can be isolated, upon treatment with ethidium bromide, that lack six major F₀ subunits, namely the nucleus-encoded subunits b, OSCP and d, and the mitochondrially encoded Atp6, 8 and 9p. Production of ρ⁰ mutants indicates that an F₁-complex carrying a mgi mutation can assemble in the absence of F₀ subunits and that suppression of ρ⁰ lethality is an intrinsic property of the altered F₁ particle. Received: 7 April 1998 / Accepted: 10 June 1998     

Radioimmunoassay for urinary metabolites of prostaglandin F2α

Antibodies against the main urinary metabolite of PGF_2α in the human, 5α,7α-dihydroxy-11-ketotetranorprosta-1,16-dioic acid, were raised in rabbits. The compound was coupled selectively in the ω position to bovine serum albumin prior to injection. The resulting antibodies did not distinguish between tetranor compounds varying only in structure at the ω carbon, and thus the assay could be used also for other metabolites of PGF_2α, e.g. the main urinary metabolite in the guinea pig, 5α,7α-dihydroxy-11-ketotetranorprostanoic acid. Labeled ligands for the assays were prepared either $\text{in vivo}$ by injection of |17,18-³H|-PGF_2α into humans after several days' treatment with indomethacin, or $\text{in vitro}$ by incubation of |17,18-³H|-15-keto-13,14-dihydro-PGF_2α with mitochondria from rat liver. The sensitivity of the assay was 10 pg or 4 pg with these two preparations, respectively.The assay was employed for a number of measurements: normal daily excretion in a number of humans; excretion of urinary metabolites during treatment with prostaglandin synthetase inhibitors in human subjects, or after intravenous injection of PGF_2α; excretion during human pregnancy; and prostaglandin production in the guinea pig during normal estrous cycles and pregnancies and after estrogen treatment.The results of these studies were in several cases compared to similar measurements earlier performed using mass spectrometric methods, and were found to agree well. Thus, this radioimmunoassay provides a simple and accurate method for estimating prostaglandin production, particularly suitable for long-term studies and for cases where repeated blood sampling must be avoided.     

Proton efflux through the chloroplast ATP synthase (CF0 · CF1) in the presence of sulfhydryl-modifying agents

The rate of photosynthetic electron transport measured in the absence of ADP and P_i is stimulated by low levels of Hg²⁺ or Ag⁺ (50% stimulation ≈ 3 Hg²⁺ or 6 Ag⁺/100 chlorophyll) to a plateau equal to the transport rate under normal phosphorylating conditions (i.e. +ADP, +P_i). Chloroplasts pretreated in the light under energizing conditions with N-ethylmaleimide show a similar stimulation of non-phosphorylating electron transport. The stimulations of non-phosphorylating electron transport by Hg²⁺, Ag⁺ and N-ethylmaleimide are reversed by the CF₁ inhibitor phlorizin, the CF₀ inhibitor triphenyltin chloride, and can be further stimulated by uncouplers such as methylamine. The Hg²⁺ and N-ethylmaleimide stimulations, but not the Ag⁺ stimulation, are completely reversed by low levels of ADP (2 μM), ATP (2 μM), and P_i (400 μM). Ag⁺, which is a potent inhibitor of ATP synthesis, has little or no effect upon phosphorylating electron transport (+ADP, +P_i). Concomitant with the stimulations of non-phosphorylating electron transport by Hg²⁺, Ag⁺ and ADP + P_i, there is a decrease in the level of membrane energization (as measured by atebrin fluorescence quenching) which is reversed when the CF₀ channel is blocked by triphenyltin. These results suggest that modification of critical CF₁ sulfhydryl residues by Hg²⁺, Ag⁺ or N-ethylmaleimide leads to the loss of intra-enzyme coupling between the transmembrane protontransferring and the ATP synthesis activities of the CF₀-CF₁ ATP synthase complex.     

Kinetic and metabolic studies of 15-methyl-prostaglandin F2α administered intra-amniotically for induction of abortion

The metabolism of tritium labeled 15-methyl-PGF_2α, administered intra-amniotically, was studied in seven cases of mid-trimester abortion. The disappearance of the compound from the amniotic sac was a very slow process. A metabolite, dinor-15-methyl-PGF_2α, was found in small amounts in the amniotic fluid and also in extracts of placenta and fetal liver, lung, and kidney. Plasma levels of 15-methyl-PGF_2α in the maternal circulation were determined.     

Effect of prostaglandin F2α on propulsive activity of the isolated segmental colon of the guinea-pig

The effects of prostaglandin F_2α (PGF_2α) on propulsive activity in segments of isolated colon and on isolated strips of guinea-pig colon were investigated.Using experimental conditions under which spontaneous propulsive activity was negligible, PGF_2α (5×10⁻⁸×1×10⁻⁶M), added to the bathing medium, increased propulsive activity in a concentration dependent manner. This increase of propulsive activity was abolished in the presence of atropine or tetrodotoxin (1×10⁻⁷g/ml).The contractions produced by PGF_2α(5×10⁻⁷ − 1×10⁻⁵M) in isolated longitudinal and circular smooth muscle strips of guinea-pig colon were unaffected in the presence of atropine or tetrodotoxin (1×10⁻⁷g/ml).From these results it is concluded that under the conditions employed in this study propulsive activity stimulated by PGF_2α may depend on the contractions of both muscle layers and stimulation of the peristalic reflex.     

The effects of prostaglandins E2 and F2α on synaptosomal accumulation and release of 3H-norepinephrine

Prostaglandins (PG) of both the E and F series may serve as modulators of norepinephrine (NE) release from peripheral sympathetic neurons. We have studied the effects of PGE₂ and PGF_2α on the accumulation and release of ³H-NE in the CNS using synaptosomes isolated from rat hypothalami.The release of ³H-NE from synaptosomes superfused with Krebs-Ringer bicarbonate buffer was multiphasic with an initial fast release phase followed by a slower release. Raising KC1 concentration of the superfusion medium to 56mM during the slow release phase is known to stimulate ³H-NE release. PGE₂ (1 × 10^?6M) attenuated ³H-NE release during the fast phase and reduced the amount of ³H-NE released due to KC1 stimulation. At lower concentrations of PGE₂ there was no change in the release profile. PGF_2α was without effect on ³H-NE release at all concentrations tested.The accumulation of ³H-NE was significantly diminished by PGE₂ at a concentration of 1 × 10^?6M, while a lower concentration (1 × 10^?7M) was ineffective. PGF_2α had no effect on ³H-NE accumulation at all concentrations investigated.     

Disappearance of prostaglandins F2α and 15-methyl F2α from amniotic fluid as measured by radioimmunoassay

A sensitive and relatively specific radioimmunoassay for 15 (S) 15 methyl prostaglandin F_2α was used to determine the levels of the drug in amniotic fluid after it had been injected intra-amniotically for termination of second trimester pregnancy. The disappearance of the free acid (tham salt) and methyl ester of the prostaglandin analogue were similar. The results of this preliminary study suggest that the drug rapidly equilibrates in the fluid and this is followed by a slow removal from the amniotic sac. A comparison with a similar study with PGF_2α, revealed that the analogue had a longer half-life in the amniotic fluid.     

Quantitative determination of prostaglandins E1 and F1α by multiple ion analysis

Quantitative assays for prostaglandins (PG) E₁ and PGF_1α are described using [3,3,4,4,5,6-²H₆]labeled prostaglandins as carriers and methyl ester-O-methyloxime-acetate (PGE₁) and methyl ester-acetate (PGF_1α) derivatives for gas - liquid chromatography/mass spectrometric analysis. Thin-layer argentation chromatography was used to separate PGE₁ from PGE₂ and 13, 14-dihydro-PGE₂. These latter compounds, which do not separate from PGE₁ using conventional thin-layer chromatography or under the gas - liquid chromatographic conditions used, can significantly interfere with the quantitative analysis of PGE₁. The method described prevents this interference and is therefore suitable for the accurate analysis of PGE₁ in biological samples containing a high concentration of PGE₂ and/or 13, 14-dihydro-PGE₂.     

Fluvoxamine rescues mitochondrial Ca transport and ATP production through σ1-receptor in hypertrophic cardiomyocytes

Aims

We previously reported that fluvoxamine, a selective serotonin reuptake inhibitor with high affinity for the σ₁-receptor (σ₁R), ameliorates cardiac hypertrophy and dysfunction via σ₁R stimulation. Although σ₁R on non-cardiomyocytes interacts with the IP₃ receptor (IP₃R) to promote mitochondrial Ca^2 + transport, little is known about its physiological and pathological relevance in cardiomyocytes.

Main methods

Here we performed Ca^2 + imaging and measured ATP production to define the role of σ₁Rs in regulating sarcoplasmic reticulum (SR)-mitochondrial Ca^2 + transport in neonatal rat ventricular cardiomyocytes treated with angiotensin II to promote hypertrophy.

Key finding

These cardiomyocytes exhibited imbalances in expression levels of σ₁R and IP₃R and impairments in both phenylephrine-induced mitochondrial Ca^2 + mobilization from the SR and ATP production. Interestingly, σ₁R stimulation with fluvoxamine rescued impaired mitochondrial Ca^2 + mobilization and ATP production, an effect abolished by treatment of cells with the σ₁R antagonist, NE-100. Under physiological conditions, fluvoxamine stimulation of σ₁Rs suppressed intracellular Ca^2 + mobilization through IP₃Rs and ryanodine receptors (RyRs). In vivo, chronic administration of fluvoxamine to TAC mice also rescued impaired ATP production.

Significance

These results suggest that σ₁R stimulation with fluvoxamine promotes SR-mitochondrial Ca^2 + transport and mitochondrial ATP production, whereas σ₁R stimulation suppresses intracellular Ca^2 + overload through IP₃Rs and RyRs. These mechanisms likely underlie in part the anti-hypertrophic and cardioprotective action of the σ₁R agonists including fluvoxamine.     

Central cardiovascular activity of prostaglandin E2, prostagladin F2∝ and prostacyclin

Prostacyclin (PGI₂), prostaglandin E₂ (PGE₂) and prostaglandin F_2∝ (PGF_2∝) were tested here in unanesthetized male Sprague-Dawley rats for their effects on the cardiovascular system as mediated by the Central nervous system. Cannulae were chronically implanted into the third cerebral ventricle, femoral arteries and femoral veins of rats. Both PGE₂ and PGF_2∝ induced increased arterial blood pressure and tachycardia by an action on the central nervous system. The changes seen with PGE₂ were larger than those observed with PGF_2∝. Only transient depressor effects were seen with PGI₂ and these changes appeared to be due to the leakage of the substance into the peripheral vascular system.     

Comparison of the effects of prostaglandins D2, F2α and E1 on spontaneous contractions of rabbit oviduct

The effects of PGD₂, PGF_2α and PGE₁ were studied on the circular muscle of post-ovulatory rabbit oviducts $\text{in vitro}$. PGE₁ inhibited spontaneous contractile activity. Lower concentrations of PGD₂ and PGF_2α were stimulatory and higher concentrations were inhibitory. Since PGD₂ may be produced in the oviduct, any hypothesis concerning the role of prostaglandins in the control of oviductal motility and ovum transport should include PGD₂ as well as PGFs and PGEs.     

Analysis of prostaglandins by electron-capture gas chromatography. I. Thermal decomposition of heptafluorobutyrate methyl esters of F1α and F2α

Triheptafluorobutyrate methyl ester (THFB-Met) derivatives are easily prepared from prostaglandins F_1α and F_2α by successive methylation and heptafluorobutyrylation. The derivatives are reasonably stable during storage, are volatile, and can be detected in the picogram range by electron-capture gas chromatography. Both derivatives exhibit peak broadening or multiple peak formation during gas chromatography at 190°–210°C. Decomposition is independent of the nature of the stationary phase and can be increased by prior heating. Studies with other derivatives suggest that thermal decomposition of the THFB-Met derivatives occuring during gas chromatography involves loss of a heptafluorobutyrate group from the allylic position 15 of the prostaglandins.     

Synthesis of prostaglandin E2 and prostaglandin F2α by toadfish red blood cells

Saline washed red blood cells of the toadfish convert [1-¹⁴C] arachidonic acid to products that cochromatograph with prostaglandin E₂ and prostaglandin F_2α. This synthesis is inhibited by indomethacin (10 μg/ml). Conversion of arachidonic acid to prostaglandin E₂ was confirmed by mass spectrometry. When saline washed toadfish red blood cells were incubated with a mixture of [1-¹⁴C]-arachidonic acid and [5,6,8,9,11,12,14,15,-³H]-arachidonic acid, comparison of the isotope ratios of the radioactive products indicated that prostaglandin F_2α was produced by reduction of prostaglandin E₂. The capacity of toadfish red blood cells to reduce prostaglandin E₂ to prostaglandin F_2α was confirmed by incubation of the cells with [1-¹⁴C] prostaglandin E₂.     

Laminaria augmentation of intra-amniotic prostaglandin F2α for the induction of mid-trimester abortion

From interpretation of 24-hour dose-response curves, it is improbable that mid-trimester abortion rates greater than about 80% can be accomplished with any one dose schedule of Prostaglandin F2α (PGF2α). To determine whether augmentation of intra-amniotic PGF2α with laminaria would improve the abortion rate, the results of a group of 22 gravidas treated with intra-amniotic PGF2α were compared to those of a group of 21 subjects treated with laminaria and an identical dose schedule of PGF2α. Patients with laminaria not only had a shorter mean abortion time (14.6 hours), but 95% aborted within 24 hours and all patients aborted within 24.5 hours of the initial PGF2α injection. Patients without laminaria had a longer mean abortion time (18.9 hours); only 68% aborted within 24 hours and one failed to abort within the 48-hour trial period. No significant differences in the frequency or severity of complications between the two groups were observed. Uterine contractility over the initial 6 hours of the induction was similar in the two groups. Therefore, augmenting the intra-amniotic PGF2α method with laminaria appears practicable.     

Intrauterine injection of prostaglandin F2α for termination of early pregnancy in out-patient

Prostaglandin F_2α administered as single intrauterine injection was used for termination of very early pregnancy in 34 out-patients. Twelve patients were induced with 1.0 mg, 18 patients with 2.0 mg and 4 patients with 4.0 mg of PGF_2α. After 2 weeks, abortion was complete in 58% induced with 1.0 mg, in 78% induced with 2.0 mg, and in all 4 patients induced with 4.0 mg. After 3 weeks, abortion was complete in 83% induced with 1.0 mg and in 100% induced with 2.0 mg. There was one failure, which occurred in the group treated by using 1.0 mg PGF_2α. Immediate side-effects were kept at an acceptable level with a very mild premedication. No serious complications occurred but curettage was performed on 2 patients because of bleeding. Spontaneous menstruation started in the 1.0 mg group within an average of 33 days (range 23–60), in the 2.0 mg group within an average of 36 days (range 25–61). The use of 2.0 mg PGF_2α for the routine termination of very early pregnancy is recommended.     

Amputation of a C-terminal helix of the γ subunit increases ATP-hydrolysis activity of cyanobacterial F1 ATP synthase

F₁ is a soluble part of F_oF₁-ATP synthase and performs a catalytic process of ATP hydrolysis and synthesis. The γ subunit, which is the rotary shaft of F₁ motor, is composed of N-terminal and C-terminal helices domains, and a protruding Rossman-fold domain located between the two major helices parts. The N-terminal and C-terminal helices domains of γ assemble into an antiparallel coiled-coil structure, and are almost embedded into the stator ring composed of α₃β₃ hexamer of the F₁ molecule. Cyanobacterial and chloroplast γ subunits harbor an inserted sequence of 30 or 39 amino acids length within the Rossman-fold domain in comparison with bacterial or mitochondrial γ. To understand the structure–function relationship of the γ subunit, we prepared a mutant F₁-ATP synthase of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, in which the γ subunit is split into N-terminal α-helix along with the inserted sequence and the remaining C-terminal part. The obtained mutant showed higher ATP-hydrolysis activities than those containing the wild-type γ. Contrary to our expectation, the complexes containing the split γ subunits were mostly devoid of the C-terminal helix. We further investigated the effect of post-assembly cleavage of the γ subunit. We demonstrate that insertion of the nick between two helices of the γ subunit imparts resistance to ADP inhibition, and the C-terminal α-helix is dispensable for ATP-hydrolysis activity and plays a crucial role in the assembly of F₁-ATP synthase.