Involvement of mitochondrial inner membrane anion carriers in the uncoupling effect of fatty acids

This paper considers stages of the search (initiated by V. P. Skulachev) for a receptor protein for fatty acids that is involved in their uncoupling effect. Based on these studies, mechanism of the ADP/ATP antiporter involvement in the uncoupling induced by fatty acids was proposed (Skulachev, V. P. (1991) FEBS Lett., 294, 158– 162). New data (suppression by carboxyatractylate of the SDS-induced uncoupling, pH-dependence of the ADP/ATP and the glutamate/aspartate antiporter contributions to the uncoupling, etc.) led to modification of this hypothesis. During discussion of the uncoupling effect of fatty acids caused by opening of the Ca²⁺-dependent pore, special attention is given to the effects of carboxyatractylate added in the presence of ADP. The functioning of the uncoupling protein UCP2 in kidney mitochondria is considered, as well as the diversity observed by us in effects of 200 µM GDP on decrease in under the influence of oleic acid added after H₂O₂ (in the presence of succinate, oligomycin, malonate). A speculative explanation of the findings is as follows: 1) products of lipid and/or fatty acid peroxidation (PPO)modify the ADP/ATP antiporter in such a way that its involvement in the fatty acid-induced uncoupling is suppressed by GDP; 2) GDP increases the PPO concentration in the matrix by suppression of efflux of fatty acid hydroperoxide anions through the UCP (Goglia, F., and Skulachev, V. P. (2003) FASEB, 17, 1585–1591)and/or of efflux of PPO anions with involvement of the GDP-sensitive ADP/ATP antiporter; 3) PPO can potentiate the oleate-induced decrease in due to inhibition of succinate oxidation.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 197–202.Original Russian Text Copyright © 2005 by Mokhova, Khailova.This revised version was published online in April 2005 with corrections to the post codes.     

Primary structure of the marmoset (Saguinus fuscicollis) hemoglobin. I. Use of tryptic maleylated peptides in the solubilization and sequence elucidation of the α- and β-chains

The primary structure of adult marmoset hemoglobin has been determined. The - and -chains of HbA were separated on a CM23 column in 8 M urea using a sodium phosphate gradient. Tryptic digests of the - and -chains were fractionated on a Dowex 50W-X2 column using a pH and pyridine acetate gradient. Large peptide fragments were obtained by the cyanogen bromide cleavage of the - and -chains, as well as by tryptic digestion of the maleylated - and -chains. The sequence was derived from the amino acid compositions and sequences of the individual tryptic peptide, automated sequence determination of intact - and -chains, as well as automated sequence determination of cyanogen bromide fragments and tryptic maleylated peptides derived from the - and -chains. The complete structure of marmoset adult hemoglobin is closely homologous to that of other primate hemoglobins. The sequence of the marmoset -chain differs from the -chain of human HbA at positions 8, 19, 23, 68, and 116. The -chain from marmoset HbA differs from the -chain of human HbA at positions 5, 13, 21, 50, 87, and 125.This work was supported in part by funds from a Physicians' Medical Education and Research Foundation Grant of the University of Tennessee Memorial Research Hospital and by NIH General Research Support Grant FR-5541 to the institution.     

Nuclear Overhauser effect investigation on GM1 ganglioside containingN-glycolyl-neuraminic acid (II3Neu5GcGgOse4Cer)

The conformational properties of the oligosaccharide chain of GM1 ganglioside containingN-glycolyl-neuraminic acid, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer, were studied through NMR nuclear Overhauser effect investigations on the monomeric ganglioside in dimethylsulfoxide, and on mixed micelles of ganglioside and dodecylphosphocholine in water. Several interresidual contacts for the trisaccharide core--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-were found to fix the relative orientitation of the three saccharides, while the glycosidic linkage of the terminal -Gal-was found to be quite mobile as the -Gal-(1-3)--GalNAc-disaccharide exists in different conformations. These results are similar to those found for two GM1 gangliosides containingN-acetyl-neuraminic acid and neuraminic acid [1].Abbreviations Ganglioside nomenclature is in accordance with Svennerholm [23] and the IUPAC-IUB Recommendations [24] GM3(Neu5Ac) II³Neu5AcLacCer, -Neu5Ac-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM3(Neu5Gc) II³Neu5GcLacCer, -Neu5Gc-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Ac) II³Neu5AcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Gc) II³Neu5GcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu) II³NeuGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu-(2-3)]--Glc-(1-1)-Cer - GD1a IV³Neu5AcII³Neu5AcGgOse₄Cer, -Neu5Ac-(2-3)--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GalNAc-GD1a IV⁴GalNAcIV³Neu5AcII³Neu5AcGgOse₄Cer, -GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - Neu neuraminic acid - Neu5Ac N-acetyl-neuraminic acid - Neu5Gc N-glycolyl-neuraminic acid - Cer ceramide     

“Mild” Uncoupling of Mitochondria

Recently, it was proposed that the thyroid hormone-mediated uncoupling in mitochondria is involved in the cellular defence system against free radicals (Skulachev V.P. (1996) Quart. Rev. Biophys. 29:169–202). This phenomenon was named mild uncoupling. It was postulated to be a protein-mediated process controlled by several factors. The data reported during the past 40 years, pointing to the protein-mediated uncoupling mechanism in mitochondria, are reviewed in a context of hypothetical properties of mild uncoupling. The mechanism of mild uncoupling is suggested to be the following: (a) mitochondria possess protein(s) that regulate the proton permeability of inner mitochondrial membrane; (b) these proteins are regulated by binding of an unidentified low-molecular-weight endogenous compound with properties resembling those of the most active artificial uncouplers like FCCP and SF6847; (c) the interaction of this compound with its target protein(s) is modulated by a thyroid hormone in a positive (i.e. enhancing the proton permeability) way and by sex steroid hormones in a negative way; (e) endogenous fatty acids can attenuate the influence of both thyroid and steroid hormones.     

Amyloid Beta Peptide Membrane Perturbation Is the Basis for its Biological Effects

Experimental studies have indicated that the mechanisms offered for explaining the neurotoxicity of amyloid beta peptide (AP) are diverse, and include altered enzyme activities, disrupted calcium homeostasis, and increased free radical formation. AP appears to interact at the cell membrane with a multitude of receptor sites and also inserts physically into the membrane matrix. This membrane insertion affects the membrane fluidity and potentially influences the function of resident membrane proteins. We propose a unifying hypothesis to explain the experimental observations of the diverse cellular responses to AP. The indiscriminate physical insertion of AP into the cell membrane unspecifically activates a host of membrane processes by perturbation of the membrane proteins. This recurrent activation of membrane processes eventually culminates in neuronal cell death. We recommend that successful therapeutic interventions should be directed at reducing or preventing the interaction of AP with neuronal cell membranes.     

Regional-scale demography of Ramonda myconi: Remnant population dynamics in a preglacial relict species

Populations of the ant Camponotus punctulatusundergo demographic explosions after agricultural activities, buildingconspicuous, vegetation-covered soil mounds. We investigated the effects ofC. punctulatus on floristic composition and soilpropertiesalong a gradient of agricultural disturbance in Northeastern Argentina. Wesampled vegetation and soil on and off anthills in,at least, three replicate plots of each of the following situations thatrepresent an increasing gradient of soil disturbance: natural grasslands, sownpastures of Digitaria decumbens, sown pastures ofSetaria sphacelata, and recently abandoned rice fields.Sets of characteristic plant species for each of the land use histories, foron and off anthills as well as for anthills ofdifferent sizes were identified through Indicator Species Analysis. 64% of thevariation in plant community composition was mainly explained by land-usehistory which was associated to the first 2 axes of a Correspondence Analysisbased on the frequency of 126 species across all sites. At the replicate scale,Correspondence Analyses revealed patterns of plant species composition relatedto the presence and size of anthills. Larger mounds became enriched in species,especially herb weeds, in comparison to smaller mounds or samples gatheredoutside the anthills. A Principal Component Analysis of soil data revealed that71% of the variation in soil properties was explained by the presence ofanthills. Soils from on anthills were more fertile than soilsfromoff anthills, independent of land-use history. The fertilityeffect of C. punctulatus mounds in addition with thevegetation patterns observed along the gradient of anthill-sizes highlights theimportance of these ants at the landscape and local scales.     

Irregular costameres represent nitric oxide synthase-1-positive sarcolemma invaginations enriched in contracted skeletal muscle fibres

NADPH diaphorase histochemistry and NOS-1 immunohistochemistry on 60 m thick frozen sections of rat extensor digitorum longus muscles led to the detection of prominent rings clearly encompassing the surface of the muscle fibres. These so far unknown costameres were usually found as doublets flanking a space of about 2 m width. Because these costameric doublets did not appear in regular periods, we designate them irregular costameres to discriminate them from regular ones with a 1 m periodicity overlying Z-discs and M-lines. Irregular costameres were thicker than the regular ones and free of intercostameres. Immunohistochemistry demonstrated that NOS-1 was co-localized with integral -dystroglycan, -sarcoglycan) and peripheral (caveolin-3, dystrophin) members of the enlarged dystrophin complex in the irregular costameres but not with non-sarcolemmal organized proteins (myosin heavy chain, -actinin, desmin and sarcoplasmic reticulum-located Ca²⁺-dependent ATPase-1). Invaginations of the sarcolemma to form irregular costameres were observed. In teased myofibres the sarcolemma between two following irregular costameres was ballooned, while the irregular costameres themselves clamped the fibres together. Finally, the number of detectable irregular costameres was significantly increased in maximally contracted extensor digitorum longus muscles generated by electric stimulation but decreased in mechanically stretched ones. Combining these observations, we hypothesize that irregular costameres belong to a reserve zone for the sarcolemma necessary for the contraction/relaxation cycle in myofibres.     

Osmotic stress-induced alterations in rice (Oryza sativa L.) and recovery on stress release

In laboratory experiments, rice plants cv Kranti were stressed osmotically using polyethylene glycol 6000 and mannitol, whereas, in pots, plants were drought stressed by withholding water supply. Both osmotic and drought stress influenced different aspects of nitrogen metabolism, resulting in a decline in the activities of aspartate aminotransferase, alanine amino-transferase and an increase in protease activity accompanied by increased free proline and alterations in other amino acid content. An influence on -amylase activity, total free sugars and starch contents was also observed, reflecting the impact of water stress on interconversion between starch and simpler sugars. Effects of polyethylene glycol 6000 as an osmotic agent were more consistent than those of mannitol during short-term (18 h) stress imposition, probably because of slight absorption of mannitol. Since the recovery for most of the parameters was substantial on release of stress, one can infer that the duration and magnitude of stress applied in the present experiments did not cause major permanent alterations in the rice cultivar Kranti. The significant basic information gathered in such experiments, particularly on recovery potential, can be utilised for varietal screening.     

Action of L-Acetylcarnitine on Different Cerebral Mitochondrial Populations from Cerebral Cortex

The maximum rate (V_max) of some mitochondrial enzymatic activities related to the energy transduction (citrate synthase, -ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, cytochrome oxidase) and amino acid metabolism (glutamate dehydrogenase, glutamate-pyruvate-transaminase, glutamate-oxaloacetate-transaminase) was evaluated in non-synaptic (free) and intra-synaptic mitochondria from rat brain cerebral cortex. Three types of mitochondria were isolated from rats subjected to i.p. treatment with L-acetylcarnitine at two different doses (30 and 60 mg·kg^–1, 28 days, 5 days/week). In control (vehicle-treated) animals, enzyme activities are differently expressed in non-synaptic mitochondria respect to intra-synaptic light and heavy ones. In fact, -ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, glutamate-pyruvate-transaminase and glutamate-oxaloacetate-transaminase are lower, while citrate synthase, cytochrome oxidase and glutamate dehydrogenase are higher in intra-synaptic mitochondria than in non-synaptic ones. This confirms that in various types of brain mitochondria a different metabolic machinery exists, due to their location in vivo. Treatment with L-acetylcarnitine decreased citrate synthase and glutamate dehydrogenase activities, while increased cytochrome oxidase and -ketoglutarate dehydrogenase activities only in intra-synaptic mitochondria. Therefore in vivo administration of L-acetylcarnitine mainly affects some specific enzyme activities, suggesting a specific molecular trigger mode of action and only of the intra-synaptic mitochondria, suggesting a specific subcellular trigger site of action.     

Variation in the karyotype of three cultivars of Narcissus tazetta L. (Amaryllidaceae)

Three cultivated varieties of Narcissus tazetta, Grand Soleil d'Or, Chinese Sacred Lily and Cypri, are triploid (2n = 3x+30) with the basic number 10. Grand Soleil d'Or has three homomorphic sets, each comprising 2 long submetacentrics, 4 long acrocentrics and 4 short acrocentrics. Karyotypes of the other two varieties are heteromorphic. Both possess one telocentric satellited chromosome. In addition, Cypri shows translocation between two chromosomes belonging to the seventh and eighth triplets. The number of secondary constrictions varies between 3 (Chinese Sacred Lily) and 4 (Grand Soleil d'Or and Cypri) which is also the number of nucleoli observed in the respective varieties.     

Mapping of Exposed Surfaces of the Nicotinic Acetylcholine Receptor by Identification of Iodinated Tyrosine Residues

Here we report on the use of iodination of the membrane-bound nicotinic acetylcholine receptor (nAChR) from Torpedo californica electric tissue in order to define surface-exposed portions of the receptor molecule. Membrane-bound nAChR was ¹²⁵I-iodinated using the oxidation agent Iodo-Gen. The iodinated subunits were separated by preparative gel electrophoresis, desalted, and cleaved with trypsin. The resulting peptides were separated by reverse-phase HPLC and the radioactive peptides were identified by mass spectrometry and protein sequencing. For the -subunit, we identified five iodinated peptides containing the tyrosine residues Tyr17, Tyr74, Tyr365, Tyr372, and Tyr428. The surface exposition of these amino acids is in agreement with the four-transmembrane-segment model (4TM model) of the nAChR, but the assignment to the intra- or extracellular surface is doubtful. According to this model, the N-terminal portion of the receptor subunits including the iodinated residues Tyr17 and Tyr74 is extracellular and Tyr372 as a site of tyrosine phosphorylation is located on the cytoplasmic side. But since this latter residue is among the first to be iodinated using an immobilized iodination agent, its true position with respect to the membrane bilayer is not clear.     

Source-sink interrelationships in lowland rice

Summary Present studies indicate that leaf could determine the degree of plants' performance and condition the level of unfilled spikelets. Environmental constraints like low light intensity itself can considerably affect the function of the leaf resulting in greater proportion of unfilled spikelets or sterility, which appears to be a post-fertilization problem. This suggests that source (leaf) is unable to cater to the requirements of the panicle. Further, the LAI studies have revealed the existence of a wide gap between spikelet production and grain filling. Panicle-tailoring studies pointed out that sterility could be reduced if panicle size is reduced. In the light of all these evidences, it was concluded that source is limiting at the present level of evolution in rice under Indian conditions.     

High irradiance response promotion of a subsequent light induction response in Sinapis alba L.

Relative quantum responsivity curves for inhibition of hypocotyl elongation in Sinapis alba L. seedlings previously grown in white light confirm that a marked end of day inhibition response can be induced by a monochromatic light treatment (30 min) at the end of the light period. In dark grown seedlings, however, no growth inhibition can be induced by a 30 min monochromatic light treatment. A prerequisite for an induction response appears to be a pretreatment with continuous light. Far red light is most effective with blue and red light showing a lesser effectiveness. The light pretreatment also shows a marked fluence rate dependency with respect to its ability to allow an induction response to manifest itself. The pretreatment required shows all the characteristics of a classical HIR response. The appearance of the effect in plants treated with the herbicide SAN 9789 seems to exclude chlorophyll as being the photoreceptor.Abbreviations SAN 9789 4-chloro-5-(methylamino)-2-(, , -trifluoro-m-tolyl)-3(2H)-pyridazinone - RG9 light long wavelength far red light (Schott RG9 colour glass) - FR far red light - WL white light - BL blue light - RL red light - D darkness - P_tot total phytochrome - P_fr far red absorbing form of phytochrome - HSR high irradiance response     

Flufenamic acid as an inducer of mitochondrial permeability transition

To assess the mechanism by which mitochondrial permeability transition (MPT) is induced by the nonpolar carboxylic acids, we investigated the effects of flufenamic acid (3-trifluoromethyl diphenylamine-2-carboxylic acid, FA) on mitochondrial respiration, electrical transmembrane potential difference (), osmotic swelling, Ca²⁺ efflux, NAD(P)H oxidation and reactive oxygen species (ROS) generation. Succinate-energized isolated rat liver mitochondria incubated in the absence or presence of 10 M Ca²⁺, 5 M ruthenium red (RR) or 1 M cyclosporin A (CsA) were used. The dose response-curves for both respiration release and dissipation were nearly linear, presenting an IC₅₀ of approximately 10 M and reaching saturation within 25-50 M, indicating that FA causes mitochondrial uncoupling by a protonophoric mechanism. Within this same concentration range FA showed the ability to induce MPT in energized mitochondria incubated with 10 M Ca²⁺, followed by dissipation and Ca²⁺ efflux, and even in deenergized mitochondria incubated with 0.5 mM Ca²⁺. ADP, Mg²⁺, trifluoperazine (TFP) and N-ethylmaleimide (NEM) reduced the extent of FA-promoted swelling in energized mitochondria by approximately one half, whereas dithiothreitol (DTT) slightly enhanced it. NAD(P)H oxidation and ROS generation (H₂O₂ production) by mitochondria were markedly stimulated by FA; these responses were partly prevented by CsA, suggesting that they may be implicated as both a cause and effect of FA-induced MPT. FA incubated with mitochondria under swelling assay conditions caused a decrease of approximately 40% in the content of protein thiol groups reacting with 5,5-dithiobis(2-nitrobenzoic acid) (DTNB). The present results are consistent with a ROS-intermediated sensitization of MPT by a direct or indirect FA interaction with inner mitochondrial membrane at a site which is in equilibrium with the NAD(P)H pool, namely thiol groups of integral membrane proteins.     

Expression of a chimeric stilbene synthase gene in transgenic wheat lines

A chimeric stilbene synthase (sts)gene was transferred into wheat. Stilbene synthases play a role in the defence against fungal diseases in some plant species (e.g. groundnut or grapevine) by producing stilbenetype phytoalexins like resveratrol. Resveratrol is also claimed to have positive effects to human health. Embryogenic scutellar calli derived from immature embryos of the two commercial German spring wheat cultivars Combi and Hanno were used as target tissue for cotransformation by microprojectile delivery. The selectable marker/reporter gene constructs contained the bargene either driven by the ubiquitinpromoter from maize (pAHC 25, also containing the uidAgene driven by the ubiquitinpromoter), or by the actinpromoter (pDM 302) from rice. The cotransferred plasmid pStil 2 consisted of a grapevine stscoding region driven by the ubiquitin promoter. Eight transgenic Combi and one Hanno T_Oplant were obtained and, except one Combi T_Oplant, found to be cotransformants due to the integration of both the stsgene and the selectable marker or reporter genes. Expression of the stsgene was proven by RTPCR, and, for the first time, by detection of the stilbene synthase product resveratrol by HPLC and mass spectrometry. The stsgene was expressed in four of the seven transgenic Combi T_{_o}plants. Two of the respective T₁progenies segregated in a Mendelian manner were still expressing the gene. Investigations into methylation of the stsgene showed that in three nonexpressing progenies inactivation was paralleled by methylation.     

Turgor regulation and cytoplasmic free Ca2+ in the algaLamprothamnium

Summary In internodal cells ofLamprothamnium succinctum, turgor regulation in response to hypotonie treatment is inhibited by lowering external Ca²⁺ concentration ([Ca²⁺]_e) from 3.9 (normal) to 0.01 (low) mM. In order to clarify whether a change in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_c) is involved in turgor regulation, the Ca²⁺ sensitive protein aequorin was injected into the cytoplasm of internodal cells. A large transient light emission was observed upon hypotonic treatment under normal [Ca²⁺]_e but not under low [Ca²⁺]_e. Thus hypotonic treatment induces a transient increase in [Ca²⁺]_c under normal [Ca²⁺]_e but not under low [Ca²⁺]_e.Abbreviations ASW artificial sea water - _i cellular osmotic pressure - [Ca²⁺]_c cytoplasmic free Ca²⁺ concentration - EDTA ethylenediamine-tetraacetic acid - EGTA ethylenglycol-bis(-aminoethyl ether(N,N-tetraacetic acid - [Ca²⁺]_e external Ca²⁺ concentration - _e external osmotic pressure - GM glass micropipette - GP glass plate - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid - MS microscope stage - OL objective lens - PIPES piperazine-N-N-bis(2-ethanesulfonic acid) - W Weight     

Defective remethylation of homocysteine is related to decreased synthesis of coenzymes B2 in thyroidectomized rats

Summary. We investigated the influence of hypothyroidism on homocysteine metabolism in rats, focusing on a hypothetical deficient synthesis of FAD by riboflavin kinases. Animals were allocated in control group (n=7), thyroidectomized rats (n=6), rats with diet deficient in vitamin B2, B9, B12, choline and methionine (n=7), thyroidectomized rats with deficient diet (n=9). Homocysteine was decreased in operated rats (2.6±1.01 vs. 4.05±1.0mol/L, P=0.02) and increased in deficient diet rats (29.56±4.52 vs. 4.05±1.0mol/L, P=0.001), when compared to control group. Erythrocyte-Glutathione-Reductase-Activation-Coefficient (index of FAD deficiency) was increased in thyroidectomized or deficient diet rats (P=0.004 for both). Methylenetetrahydrofolate-reductase and methionine-synthase activities were decreased in thyroidectomized rats but not in those subjected to deficient diet. Cystathionine--synthase was increased only in operated rats. Taken together, these results showed a defective re-methylation in surgical hypothyroidism, which was due in part to a defective synthesis of vitamin B2 coenzymes. This defective pathway was overcompensated by the increased Cystathionine--synthase activity.     

Chloramine-T alters the nerve membrane birefringence response

Summary The change in birefringence during depolarizing voltage-clamp pulses of internally perfused squid giant axons are biphasic. There is a rapid decrease in birefringence with a 220-sec half time at 8°C followed by a slow decrease over the next several milliseconds. After the pulse there is a rapid recovery which is smaller than the initial rapid decrease followed by a slow recovery phase. The rate of change of the slow phase during the pulse is more rapid for larger depolarizations. After the pulse the rate of change is more rapid for more negative potentials.3.6mm chloramine-T, applied externally until the sodium currents were prolonged and inactivation was removed, removed the slow phase of the birefringence response both during and after the pulse and made the fast off response as large as the fast on response. Two anesthetics reduced the birefringence response by about 20%.A rocking helix model is presented which relates the birefringence findings and earlier gating current experiments.     

Changes in the permeability of the inner mitochondrial membrane associated with plastid development

Summary Mitochondria isolated from greening etiolated laminae of Avena sativa L. show changes in the permeability of their inner membranes during chloroplast development similar to those described earlier for plastids. Oxalo-acetate, succinate and -keto-glutarate permeate most readily inner membranes of mitochondria isolated from laminae given 2 h illumination whilst glutamate and glycine show later and more general penetration into the matrix spaces of mitochondria from greening tissue. Aminolevulinic acid (ALA) by contrast does not readily enter Avena mitochondria especially those isolated from laminae illuminated for longer than 2 h.Abbreviations ALA amino-levulinic acid - HEPES N-2-hydroxyethyl-piperazine-N-2-ethane-sulphonic acid     

Role of luminal buffers in renal tubular acidification

Summary The acidification kinetics of artificial solutions containing buffers of different permeancy were studied in rat proximal tubules by means of stationary microperfusion techniques. Luminal pH changes were measured by antimony microelectrodes and used to calculate net rates of acidification and the approach to steady-state pH levels. For most buffer species, tracer efflux out of the lumen was compared with changes in buffer concentration as derived from calculations based on the Henderson Hasselbalch equation. Steady-state luminal pH was similar for most buffer systems studied. However, secretory hydrogen ion fluxes into the lumen were significantly higher for permeant than for less permeant buffers. The most likely explanation is that permeant buffers behave as open systems maintaining constant low diffusible acid levels in the lumen, whereas impermeant buffers behave as closed systems in which nonionized acid levels are maintained at higher levels. A behavior consistent with this thesis was directly demonstrated for glycodiazine and, to a lesser degree, for DMO. In contrast, phosphate and creatinine behave like buffers in a closed cystem. Characteristics of proximal tubular acidification, of buffer reabsorption, and the effect thereupon of carbonic anhydrase inhibitors are satisfactorily explained by an essential role of (1) hydrogen ion secretion, (2) pK differences, and (3) different permeance of the non-ionized buffer species. However, specific transport mechanisms may, in addition, also contribute to differences in transepithelial buffer movement.