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.     

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.     

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.     

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.     

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.     

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.     

Radioimmunoassays of prostaglandin F2α and prostaglandin F2α-main urinary metabolite with prostaglandin-125I-tyrosine methylester amide

Radioimmunoassays for measuring prostaglandin F_2α (PGF_2α) and 5α, 7α-dihydroxy-11-keto tetranorprosta-1,16-dioic acid, PGF_2α-main urinary metabolite (PGF_2α-MUM), with ¹²⁵I-tyrosine methylester amide (TMA) of PGF_2α and PGF_2α-MUM were developed.Antibody to PGF_2α was produced in rabbits immunized with conjugates of PGF_2α coupled to bovine serum albumine. Antibody to PGF_2α-MUM was also produced in rabbits immunized with conjugates of PGF_2α-MUM coupled to bovine serum albumin.PGF_2α-¹²⁵I-TMA had an affinity to antiserum to PGF_2α. PGF_2α-MUM-¹²⁵I-TMA also responded to antiserum to PGF_2α-MUM.     

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.     

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.     

Induction of abortion by intra- and extra-amniotic prostaglandin F2α administration

Legal abortion was induced by intrauterine administration of prostaglandin F_2α in 115 patients during the 11th – 20th week of pregnancy. An intra-amniotic method was used in 61 of the cases, an extra-amniotic one in 54 cases. The average total dose administered was 35.1 mg (range 5 – 65 mg) in the intra-amniotic group, and 6358 μg (range 1500 – 14000 μg) in the extra-amniotic group. Abortion rate was 92 % in the intra-amniotic material and 72 % in the extra-amniotic material, and side-effects, mainly gastrointestinal irritation, were noted in 74 % of the intra-amniotic cases and 54 % of the extra-amniotic ones. If total doses of 4750 μg or more were administered in the extra-amniotic cases, abortion rate went up to 80 %, but the frequency of side-effects simultaneously increased to 64 %. No serious complications occurred. Intrauterine prostaglandin induction is well suited therapeutic abortions in the second trimester, and the intra-amniotic technique is more practicable than the extra-amniotic one. The latter is applicable in cases where the puncture of the amniotic cavity is difficult to achieve, e.g. in cases of fetus mortuus and hydatiform mole.     

Determination of formation constants for complexes of very high stability: log β4 for the [Pd(CN)4] ion

The formation constant, log β₄ = 62.3 for [Pd(CN)₄]²⁻ is reported at 25 °C in 0.1 M NaClO₄. This value of log β₄ was determined using a competition reaction, monitored using UV-Vis spectroscopy and ¹H NMR. The competition reaction used was with the tetraamine ligand 2,3,2-tet(1,4,8,11-tetraazaundecane), for which log K₁ = 47.8 at 25 °C in 0.1 M NaClO₄ was determined by competition with thiocyanate, as described by earlier workers (Q.Y. Yan, G. Anderegg, Inorg. Chim. Acta 105 (1985) 121.). Also reported is a value of log β₄ for the [Pd(SCN)₄]²⁻ ion of 27.2 in 0.1 M NaClO₄, determined by competition with 2,2,2-tet. Measurement of log K₁ for cyclam with Pd(II) was attempted using a competition reaction with cyanide, combined with the very high value of log β₄ for [Pd(CN)₄]²⁻ measured here. It appeared that the equilibrium being followed was actually [Pd(cyclam)]²⁺ + 2CN⁻ ? [Pd(cyclam)(CN)₂], for which a constant of log K = 5.2 was obtained. ¹H NMR and IR studies suggested that the complex [Pd(cyclam)(CN)₂] was prone to oxidation to Pd(IV), followed by disproportionation to [Pd(cyclam)]²⁺ and, presumably, (CN)₂. The very high value of log β₄ for [Pd(CN)₄]²⁻ found here appears to be the highest formation constant known for any metal ion.     

Comparison of the actions of the 15-methyl analogs of prostaglandins E2 and F2α on hormone levels after their administration for therapeutic abortion

Plasma levels of progesterone, total estrogens and HCG were measured after the administration of 15 (S) 15-methyl prostaglandin E₂ methyl ester (15-methyl PGE₂) or 15 (S) 15-methyl prostaglandin F_2α free acid (15-methyl PGF_2α) for therapeutic abortion during the first trimester of pregnancy. 15-methyl PGE₂ given intramuscular (im) in a dose of 50μg resulted in the termination in pregnancy in four out of five patients; these subjects exhibited falls in hormone concentrations. However, an im injection of 500μg 15-methyl PGF_2α did not affect the hormone levels nor did it produce abortion in any of the five subjects studied. The results confirm that 15-methyl PGE₂ is a potent abortifacient and this action may be related to an effect that this compound has on hormone production by the corpus luteum or the feto-placental unit; 15-methyl PGF_2α does not share the same action in the dose range investigated.     

Levels of prostaglandin F2α in amniotic fluid during pregnancy and labour

Levels of prostaglandin F_2α (PGF_2α) in the amniotic fluid were determined by radioimmunoassay. Concentrations of the prostaglandin were relatively constant between 15 and 35 weeks' gestation, but an increase was observed after 36 weeks. The rise was continued up to 44 weeks. A still greater elevation of PGF_2α levels was recorded during labour, when the levels were related to the amount of cervical dilatation.Amniotic fluid PGF_2α levels in toxaemia of pregnancy did not significantly differ from those found in normal pregnancy.     

Mass fragmentographic determination of prostaglandin F2α in human and rabbit urine

Analysis of prostaglandin F_2α (PGF_2α) in urine is a useful indicator of renal prostaglandin synthesis. A mass fragmentographic method for PGF_2α analysis in human urine was developed using [3,3,4,4-²H₄]PGF_2α as an internal standard and carrier. PGF_2α was extracted from urine (20 ml) with chloroform, purified by preparative thin-layer chromatography and converted to the methyl ester trimethylsilyl ether before analysis by gas chromatograph—mass spectrometry. The specificity of the urine analysis was demonstrated by retention time and the use of two pairs of fragments m/e 494/498 and 513/517 with the same results. The coefficient of variation for duplicate analysis averaged 12.6%, n = 17. Urine from recumbent women contained 4.9 ± 2.6 (S.D.) ng/ml or 4.1 ± 1.0 ng PGF_2α per mg creatinine (n = 10) with little diurnal variation. Male urine contained 5.0 ± 2.7 (S.D.) ng/ml or 3.7 ± 2.1 ng/mg creatinine (n = 10). Similar concentrations were found in boys and in girls. These observations indicate that urinary PGF_2α originates from the kidneys with little contribution from the male accessory sexual glands. This method can also be applied to analysis of PGF_2α in rabbit urine.