Inhalation of molecular hydrogen increases breath acetone excretion during submaximal exercise: a randomized, single-blinded, placebo-controlled study.

Aerobic exercise is widely accepted as a beneficial option for reducing fat in humans. Recently, it has been suggested that molecular hydrogen (H2) augments mitochondrial oxidative phosphorylation. Therefore, the hypothesis that inhaling H2 could facilitate lipid metabolism during aerobic exercise was investigated in the current study by measuring the breath acetone levels, which could be used as non-invasive indicators of lipid metabolism. This study aimed to investigate the effect of inhaling H2 on breath acetone output during submaximal exercise using a randomized, single-blinded, placebo-controlled, and cross-over experimental design. After taking a 20-minute baseline measurement, breath acetone levels were measured in ten male subjects who performed a 60% peak oxygen uptake-intensity cycling exercise for 20 minutes while inhaling either 1% H2 or a control gas. In another experiment, six male subjects remained in a sitting position for 45 minutes while inhaling either 1% H2 or a control gas. H2 significantly augmented breath acetone and enhanced oxygen uptake during exercise (P < 0.01). However, it did not significantly change oxidative stress or antioxidant activity responses to exercise, nor did it significantly alter the breath acetone or oxygen uptake during prolonged resting states. These results suggest that inhaling H2 gas promotes an exercise-induced increase in hepatic lipid metabolism. The study was approved by the Ethical Committee of Chubu University, Japan (approved No. 260086-2) on March 29, 2018.

Acid-Base Basics.

Although students initially learn of ionic buffering in basic chemistry, buffering and acid-base transport in biology often is relegated to specialized classes, discussions, or situations. That said, for physiology, nephrology, pulmonology, and anesthesiology, these basic principles often are critically important for mechanistic understanding, medical treatments, and assessing therapy effectiveness. This short introductory perspective focuses on basic chemistry and transport of buffers and acid-base equivalents, provides an outline of basic science acid-base concepts, tools used to monitor intracellular pH, model cellular responses to pH buffer changes, and the more recent development and use of genetically encoded pH-indicators. Examples of newer genetically encoded pH-indicators (pHerry and pHire) are provided, and their use for in vitro, ex vivo, and in vivo experiments are described. The continued use and development of these basic tools provide increasing opportunities for both basic and potentially clinical investigations.

Effects of Post-Treatment Hydrogen Gas Inhalation on Uveitis Induced by Endotoxin in Rats.

BACKGROUND Molecular hydrogen (H2) has been widely reported to have benefiicial effects in diverse animal models and human disease through reduction of oxidative stress and inflammation. The aim of this study was to investigate whether hydrogen gas could ameliorate endotoxin-induced uveitis (EIU) in rats. MATERIAL AND METHODS Male Sprague-Dawley rats were divided into a normal group, a model group, a nitrogen-oxygen (N-O) group, and a hydrogen-oxygen (H-O) group. EIU was induced in rats of the latter 3 groups by injection of lipopolysaccharide (LPS). After that, rats in the N-O group inhaled a gas mixture of 67% N2 and 33% O2, while those in the H-O group inhaled a gas mixture of 67% H2 and 33% O2. All rats were graded according to the signs of uveitis after electroretinography (ERG) examination. Protein concentration in the aqueous humor (AqH) was measured. Furthermore, hematoxylin-eosin staining and immunostaining of anti-ionized calcium-binding adapter molecule 1 (Iba1) in the iris and ciliary body (ICB) were carried out. RESULTS No statistically significant differences existed in the graded score of uveitis and the b-wave peak time in the Dark-adapted 3.0 ERG among the model, N-O, and H-O groups (P>0.05), while rats of the H-O group showed a lower concentration of AqH protein than that of the model or N-O group (P<0.05). The number of the infiltrating cells in the ICB of rats from the H-O group was not significantly different from that of the model or N-O group (P>0.05), while the activation of microglia cells in the H-O group was somewhat reduced (P<0.05). CONCLUSIONS Post-treatment hydrogen gas inhalation did not ameliorate the clinical signs, or reduce the infiltrating cells of EIU. However, it inhibited the elevation of protein in the AqH and reduced the microglia activation.

[Protective effects of hydrogen-rich medium on lipopolysaccharides-induced injury in human periodontal ligament cells].

OBJECTIVE: In this study, lipopolysaccharides (LPS) was used to damage human periodontal ligament cells (hPDLCs) and consequently investigate the protective effects of hydrogen on reducing oxidative stress and cell apoptosis rate. METHODS: hPDLCs were isolated, and then cultured with normal medium+1 µg·mL⁻¹ LPS or with hydrogen-rich medium+
1 µg·mL⁻¹ LPS. Cell proliferation activity was assessed using a cell counting kit-8 (CCK-8), and lactic dehydrogenase (LDH) release was also detected. The activities of superoxide dismutase (SOD) and catalase (CAT), and the level of malonaldehyde (MDA) in supernatants were also measured. Cell apoptosis was detected by flow cytometry at 24 h after LPS stimulation. RESULTS: CCK-8 results showed that hydrogen could significantly improve hPDLCs growth and decrease cell apoptosis under LPS stimulation (P<0.05). However, no significant difference in LDH release was found between the two groups. The CAT levels significantly increased at 6 and 12 h in the hydrogen-rich medium as compared with the normal medium group (P<0.05, P<0.01, respectively). However, SOD levels were not significant different at each time point. At 6 h after LPS stimulation, the MDA levels in the cell supernatant of hydrogen-rich medium group were significantly reduced as compared with those in the normal medium group (P<0.05). CONCLUSIONS: The hydrogen-rich medium can effectively improve hPDLCs proliferation activity and antioxidant capacity and reduce apoptosis and oxidative stress under LPS stimulation.

Arabidopsis plasma membrane H+-ATPase genes AHA2 and AHA7 have distinct and overlapping roles in the modulation of root tip H+ efflux in response to low-phosphorus stress.

Phosphorus deficiency in soil is one of the major limiting factors for plant growth. Plasma membrane H+-ATPase (PM H+-ATPase) plays an important role in the plant response to low-phosphorus stress (LP). However, few details are known regarding the action of PM H+-ATPase in mediating root proton (H+) flux and root growth under LP. In this study, we investigated the involvement and function of different Arabidopsis PM H+-ATPase genes in root H+ flux in response to LP. First, we examined the expressions of all Arabidopsis PM H+-ATPase gene family members (AHA1-AHA11) under LP. Expression of AHA2 and AHA7 in roots was enhanced under this condition. When the two genes were deficient in their respective Arabidopsis mutant plants, root growth and responses of the mutants to LP were highly inhibited compared with the wild-type plant. AHA2-deficient plants exhibited reduced primary root elongation and lower H+ efflux in the root elongation zone. AHA7-deficient plants exhibited reduced root hair density and lower H+ efflux in the root hair zone. The modulation of H+ efflux by AHA2 or AHA7 was affected by the action of 14-3-3 proteins and/or auxin regulatory pathways in the context of root growth and response to LP. Our results suggest that under LP conditions, AHA2 acts mainly to modulate primary root elongation by mediating H+ efflux in the root elongation zone, whereas AHA7 plays an important role in root hair formation by mediating H+ efflux in the root hair zone.

Na+ /H+ exchange via the Drosophila vesicular glutamate transporter mediates activity-induced acid efflux from presynaptic terminals.

KEY POINTS: Intracellular pH regulation is vital to neurons as nerve activity produces large and rapid acid loads in presynaptic terminals. Rapid clearance of acid loads is necessary to maintain control of neurotransmission, but neuronal acid clearance mechanisms remain poorly understood. Glutamate is loaded into synaptic vesicles via the vesicular glutamate transporter (VGLUT), a mechanism conserved across phyla, and this study reports a previously unknown role for VGLUT as an acid-extruding protein when deposited in the plasmamembrane during exocytosis. The finding was made in Drosophila (fruit fly) larval motor neurons through a combined pharamacological and genetic dissection of presynaptic pH homeostatic mechanisms. A dual role for VGLUT serves to integrate neuronal activity and pH regulation in presynaptic nerve terminals. ABSTRACT: Neuronal activity can result in transient acidification of presynaptic terminals, and such shifts in cytosolic pH (pHcyto ) probably influence mechanisms underlying forms of synaptic plasticity with a presynaptic locus. As neuronal activity drives acid loading in presynaptic terminals, we hypothesized that the same activity might drive acid efflux mechanisms to maintain pHcyto homeostasis. To better understand the integration of neuronal activity and pHcyto regulation we investigated the acid extrusion mechanisms at Drosophila glutamatergic motorneuron terminals. Expression of a fluorescent genetically encoded pH indicator, named 'pHerry', in the presynaptic cytosol revealed acid efflux following nerve activity to be greater than that predicted from measurements of the intrinsic rate of acid efflux. Analysis of activity-induced acid transients in terminals deficient in either endocytosis or exocytosis revealed an acid efflux mechanism reliant upon synaptic vesicle exocytosis. Pharmacological and genetic dissection in situ and in a heterologous expression system indicate that this acid efflux is mediated by conventional plasmamembrane acid transporters, and also by previously unrecognized intrinsic H+ /Na+ exchange via the Drosophila vesicular glutamate transporter (DVGLUT). DVGLUT functions not only as a vesicular glutamate transporter but also serves as an acid-extruding protein when deposited on the plasmamembrane.

Hierarchical spike clustering analysis for investigation of interneuron heterogeneity.

Action potentials represent the output of a neuron. Especially interneurons display a variety of discharge patterns ranging from regular action potential firing to prominent spike clustering or stuttering. The mechanisms underlying this heterogeneity remain incompletely understood. We established hierarchical cluster analysis of spike trains as a measure of spike clustering. A clustering index was calculated from action potential trains recorded in the whole-cell patch clamp configuration from hippocampal (CA1, stratum radiatum) and entorhinal (medial entorhinal cortex, layer 2) interneurons in acute slices and simulated data. Prominent, region-dependent, but also variable spike clustering was detected using this measure. Further analysis revealed a strong positive correlation between spike clustering and membrane potentials oscillations but an inverse correlation with neuronal resonance. Furthermore, clustering was more pronounced when the balance between fast-activating K(+) currents, assessed by the spike repolarisation time, and hyperpolarization-activated currents, gauged by the size of the sag potential, was shifted in favour of fast K(+) currents. Simulations of spike clustering confirmed that variable ratios of fast K(+) and hyperpolarization-activated currents could underlie different degrees of spike clustering and could thus be crucial for temporally structuring interneuron spike output.

Hydrogen-rich water regulates cucumber adventitious root development in a heme oxygenase-1/carbon monoxide-dependent manner.

Hydrogen gas (H2) is an endogenous gaseous molecule in plants. Although its reputation is as a "biologically inert gas", recent results suggested that H2 has therapeutic antioxidant properties in animals and plays fundamental roles in plant responses to environmental stresses. However, whether H2 regulates root morphological patterns is largely unknown. In this report, hydrogen-rich water (HRW) was used to characterize H2 physiological roles and possible signaling transduction pathways in the promotion of adventitious root (AR) formation in cucumber explants. Our results showed that a 50% concentration of HRW was able to mimic the effect of hemin, an inducer of a carbon monoxide (CO) synthetic enzyme, and heme oxygenase-1 (HO-1), in restoring AR formation in comparison with the inhibition effect conferred by auxin-depletion treatment alone. It was further shown that the inducible effect of HRW could be further blocked by the co-treatment with N-1-naphthylphtalamic acid (NPA; an auxin transport inhibitor). The HRW-induced response, at least partially, was HO-1-dependent. This conclusion was supported by the fact that the exposure of cucumber explants to HRW up-regulates cucumber HO-1 gene expression and its protein levels. HRW-mediated induction of representative target genes related to auxin signaling and AR formation, such as CsDNAJ-1, CsCDPK1/5, CsCDC6, CsAUX22B-like, and CsAUX22D-like, and thereafter AR formation (particularly in the AR length) was differentially sensitive to the HO-1 inhibitor zinc protoporphyrin IX (ZnPP). Above blocking actions were clearly reversed by CO, further confirming that the above response was HO-1/CO-specific. However, the addition of a well-known antioxidant, ascorbic acid (AsA), failed to influence AR formation triggered by HRW, thus ruling out the involvement of redox homeostasis in this process. Together, these results indicated that HRW-induced adventitious rooting is, at least partially, correlated with the HO-1/CO-mediated responses. We also suggested that exogenous HRW treatment on plants might be a good option to induce root organogenesis.

[Raman spectroscopy study of the effect of H+ on the oxygen affinity capacity of hemoglobin].

The hemoglobin was extracted from the blood which was provided by the healthy volunteers and the impact of the pH on hemoglobin oxygen binding capacity was studied with microscopic Raman spectroscopy. The results indicated that: under the excitation light of 514.5 nm, with the reducing of the oxygen partial pressure (PO2), the Raman peak intensity at 1 375, 1 562, 1 585 and 1 638 cm(-1) of the control hemoglobin (pH 7.4) reduced gradually, among which, the change of the 1 375 and 1 638 cm(-1) were the most significant and had a good relevance with the PO2. The curves were plotted by regarding the PO2 as the x-axis and the Raman absolute intensity as the y-axis, and the relationship between hemoglobin Raman absolute intensity of the 1 375 and 1 638 cm(-1) and their related PO2 levels when the pH was 5.7, 7.4 and 8.0 respectively were analyzed. The data was well linear fitted and the fitting equation was obtained. The relationship of the slope (Raman intensity/PO2 level) among them were K8.0 > K7.4 > K5.7, indicating that the lower the pH, the easier the release of the oxygen molecules. It was showed that the Raman spectroscopy technique could be used to detect the oxygen binding rate of hemoglobin quantitatively, and the effect of the PH on oxygen binding state of hemoglobin could be observed, which could provide a new method and make a foundation for the monitoring of the PO2 levels in the blood, as well as the research on the regulatory factors of the blood oxygen affinity, such as H+ and CO2.

Hydrogen tunneling links protein dynamics to enzyme catalysis.

The relationship between protein dynamics and function is a subject of considerable contemporary interest. Although protein motions are frequently observed during ligand binding and release steps, the contribution of protein motions to the catalysis of bond making/breaking processes is more difficult to probe and verify. Here, we show how the quantum mechanical hydrogen tunneling associated with enzymatic C-H bond cleavage provides a unique window into the necessity of protein dynamics for achieving optimal catalysis. Experimental findings support a hierarchy of thermodynamically equilibrated motions that control the H-donor and -acceptor distance and active-site electrostatics, creating an ensemble of conformations suitable for H-tunneling. A possible extension of this view to methyl transfer and other catalyzed reactions is also presented. The impact of understanding these dynamics on the conceptual framework for enzyme activity, inhibitor/drug design, and biomimetic catalyst design is likely to be substantial.

Recent advances in hydrogen research as a therapeutic medical gas.

Recent basic and clinical research has revealed that hydrogen is an important physiological regulatory factor with antioxidant, anti-inflammatory and anti-apoptotic protective effects on cells and organs. Therapeutic hydrogen has been applied by different delivery methods including straightforward inhalation, drinking hydrogen dissolved in water and injection with hydrogen-saturated saline. This review summarizes currently available data regarding the protective role of hydrogen, provides an outline of recent advances in research on the use of hydrogen as a therapeutic medical gas in diverse models of disease and discusses the feasibility of hydrogen as a therapeutic strategy. It is not an overstatement to say that hydrogen's impact on therapeutic and preventive medicine could be enormous in the future.

A biohydrogen fuel cell using a conductive polymer nanocomposite based anode.

This paper introduces a newly designed biohydrogen fuel cell by integrating a bioreactor for hydrogen production with the anode chamber in a hydrogen fuel cell. Two different composites of platinum nanoparticles decorated on functionalised multi-walled carbon nanotubes (Pt/fMWCNTs) and polyaniline (PANI) were fabricated using the electrochemical polymerisation method and used as anodes. The biohydrogen fuel cell using a thin film of PANI nanofibres deposited on Pt/fMWCNTs/carbon paper as the anode showed much higher power density than the cell using a core-shell structure PANI/Pt/fMWCNTs and Pt/fMWCNTs without PANI based anodes. The structural differences between these two composites and their effects on the interaction with hydrogen gas inside the anode chamber leading to the difference in power density of the fuel cell were also discussed. The maximum power density was 613.5 mW m(-2), which was obtained at a current density of about 2.55 A m(-2) with a cell voltage of 0.24 V using 20 mL single-chamber air-cathode, compact biohydrogen fuel cell.

Physiological and pharmacokinetic roles of H+/organic cation antiporters (MATE/SLC47A).

Vectorial secretion of cationic compounds across tubular epithelial cells is an important function of the kidney. This uni-directed transport is mediated by two cooperative functions, which are membrane potential-dependent organic cation transporters at the basolateral membranes and H+/organic cation antiporters at the brush-border membranes. More than 10 years ago, the basolateral organic cation transporters (OCT1-3/SLC22A1-3) were isolated, and molecular understandings for the basolateral entry of cationic drugs have been greatly advanced. However, the molecular nature of H+/organic cation antiport systems remains unclear. Recently, mammalian orthologues of the multidrug and toxin extrusion (MATE) family of bacteria have been isolated and clarified to function as H+/organic cation antiporters. In this commentary, the molecular characteristics and pharmacokinetic roles of mammalian MATEs are critically overviewed focusing on the renal secretion of cationic drugs.

Primary radical pair P(+)H(-) lifetime in rhodobacter sphaeroides with blocked electron transfer to Q(A). Effect of o-phenanthroline.

Transient absorption spectroscopy with a time resolution of approximately 1 ns was applied to study the decay of the primary radical pair P+H- in Rhodobacter sphaeroides R-26 reaction centers with blocked electron transfer from H- to QA. The block in the electron transfer was realized in two ways: by either reducing or removing QA. We found very different kinetics of the P+H- decay in these two cases. Convolution of the multiexponential decay with the instrument response function allowed resolution of as many as three kinetic components of <1-, 3-4-, and 9-12-ns lifetimes in chromatophores with QA reduced and in isolated reaction centers both with QA either reduced or removed (with or without o-phenanthroline) but with variable relative amplitudes. Removing QA or adding o-phenanthroline to isolated reaction centers increased the amplitude of the slowest decay phase relative to that of the fastest phase. On the basis of these observations, we propose that reaction centers adopt three conformational states characterized by different decay kinetics of P+H-. These conformational states appear to be controlled by the charges in the vicinity of the QA site as revealed by the effects of QA reduction and o-phenanthroline-mediated protonation of the sites close to QA.

Hydrogen symbioses in evolution and disease.

Hydrogen is the source of energy that unites the metabolisms and fuels the innovative potentials of all living organisms. Autotrophs use hydrogen emitted into hydrothermal vents, where symbiotic communities that share hydrogen thrive. On the surface, life developed using photons to cleave water, releasing hydrogen carried into a reverse Krebs cycle to produce carbohydrates, from which hydrogen and its constituent electron and proton are extracted. Fluctuant electrogenic power is harnessed by extensive exchanges and symbiotic sharing schemes of hydrogen sources and carriers. These communicate with electrostatic nuclear centres, forming a positive feedback loop. If the proton-motive circuitry fails from loss of Redox potential, premature ageing and all-category disease can result.

Oppositely directed H+ gradient functions as a driving force of rat H+/organic cation antiporter MATE1.

Recently, we have isolated the rat (r) H(+)/organic cation antiporter multidrug and toxin extrusion 1 (MATE1) and reported its tissue distribution and transport characteristics. Functional characterization suggested that an oppositely directed H(+) gradient serves as a driving force for the transport of a prototypical organic cation, tetraethylammonium, by MATE1, but there is no direct evidence to prove this. In the present study, therefore, we elucidated the driving force of tetraethylammonium transport via rMATE1 using plasma membrane vesicles isolated from HEK293 cells stably expressing rMATE1 (HEK-rMATE1 cells). A 70-kDa rMATE1 protein was confirmed to exist in HEK-rMATE1 cells, and the transport of various organic cations including [(14)C]tetraethylammonium was stimulated in intracellular acidified HEK-rMATE1 cells but not mock cells. The transport of [(14)C]tetraethylammonium in membrane vesicles from HEK-rMATE1 cells exhibited the overshoot phenomenon only when there was an outwardly directed H(+) gradient, as observed in rat renal brush-border membrane vesicles. The overshoot phenomenon was not observed in the vesicles from mock cells. The stimulated [(14)C]tetraethylammonium uptake by an H(+) gradient [intravesicular H(+) concentration ([H(+)](in)) > extravesicular H(+) concentration ([H(+)](out))] was significantly reduced in the presence of a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). [(14)C]tetraethylammonium uptake was not changed in the presence of valinomycin-induced membrane potential. These findings definitively indicate that an oppositely directed H(+) gradient serves as a driving force of tetraethylammonium transport via rMATE1, and this is the first demonstration to identify the driving force of the MATE family. The present experimental strategy is very useful in identifying the driving force of cloned transporters whose driving force has not been evaluated.

The surface energy of water; molecular biology of the working mechanisms.

The surface tension and surface energy of water, have been studied for two centuries. The ability to do physical chemical work dates from the decades at the close of the nineteenth century and opening of the twentieth. Teaching and research popularity dropped in the post-war years, and today is practically unique. Before 1846, Laplace, the French scientist, found that pressure was needed to force water, or other liquid, through small holes. The pressure needed was greater for smaller holes, and the relationship depended upon the surface tension of the liquid under test, and the inverse of the radius of the holes. The Laplace formula still appears in Physiological textbooks. The importance in the small air tubes of respiratory physiology, and in the capillary circulation generally, is still studied. The use of surface energy for protein conformational changes has received little attention. A proposed scheme for the human erythrocyte glucose transporter, although shown to be experimentally consistent, has been disregarded. A possible reason is the difficulty of comprehending how surface energy can actually perform mechanical work. Therefore, studies with molecular models, which increase the understanding of how mechanical work is done are described in more detail here.

Mechanism of acid-induced bone resorption.

PURPOSE OF REVIEW: This review presents our current understanding of the way metabolic acidosis induces calcium efflux from bone, and in the process, buffers additional systemic hydrogen ions associated with acidosis. RECENT FINDINGS: Acid-induced changes in bone mineral are consistent with a role for bone as a proton buffer. In response to metabolic acidosis in an in-vitro bone organ culture system, we observed a fall in mineral sodium, potassium, carbonate and phosphate, which each buffer protons and in vivo should increase systemic pH towards the physiologic normal. Initially, metabolic acidosis stimulates physicochemical mineral dissolution and subsequently cell-mediated bone resorption. Acidosis suppresses the activity of bone-resorbing cells, osteoblasts, decreasing gene expression of specific matrix proteins and alkaline phosphatase activity. There is concomitant acid stimulation of prostaglandin production by osteoblasts, which acting in a paracrine manner increases synthesis of the osteoblastic receptor activator of nuclear factor kappa B ligand (RANKL). The acid induction of RANKL then stimulates osteoclastic activity and recruitment of new osteoclasts to promote bone resorption and buffering of the proton load. Both the regulation of RANKL and acid-induced calcium efflux from bone are mediated by prostaglandins. SUMMARY: Metabolic acidosis, which occurs during renal failure, renal insufficiency or renal tubular acidosis, results in decreased systemic pH and is associated with an increase in urine calcium excretion. The apparent protective function of bone to help maintain systemic pH, which has a clear survival advantage for mammals, will come partly at the expense of its mineral stores.

A carrier-mediated mechanism for pyridoxine uptake by human intestinal epithelial Caco-2 cells: regulation by a PKA-mediated pathway.

Vitamin B6 is essential for cellular functions and growth due to its involvement in important metabolic reactions. Humans and other mammals cannot synthesize vitamin B6 and thus must obtain this micronutrient from exogenous sources via intestinal absorption. The intestine, therefore, plays a central role in maintaining and regulating normal vitamin B6 homeostasis. Due to the water-soluble nature of vitamin B6 and the demonstration that transport of other water-soluble vitamins in intestinal epithelial cells involves specialized carrier-mediated mechanisms, we hypothesized that transport of vitamin B6 in these cells is also carrier mediated in nature. To test this hypothesis, we examined pyridoxine transport in a model system for human enterocytes, the human-derived intestinal epithelial Caco-2 cells. The results showed pyridoxine uptake to be 1) linear with time for up to 10 min of incubation and to occur with minimal metabolic alteration in the transported substrate, 2) temperature and energy dependent but Na+ independent, 3) pH dependent with higher uptake at acidic compared with alkaline pHs, 4) saturable as a function of concentration (at buffer pH 5.5 but not 7.4) with an apparent Michaelis-Menten constant (Km) of 11.99 +/- 1.41 microM and a maximal velocity (Vmax) of 67.63 +/- 3.87 pmol. mg protein-1. 3 min-1, 5) inhibited by pyridoxine structural analogs (at buffer pH 5.5 but not 7.4) but not by unrelated compounds, and 6) inhibited in a competitive manner by amiloride with an apparent inhibitor constant (Ki) of 0.39 mM. We also examined the possible regulation of pyridoxine uptake by specific intracellular regulatory pathways. The results showed that whereas modulators of PKC, Ca+2/calmodulin (CaM), and nitric oxide (NO)-mediated pathways had no effect on pyridoxine uptake, modulators of PKA-mediated pathway were found to cause significant reduction in pyridoxine uptake. This reduction was mediated via a significant inhibition in the Vmax, but not the apparent Km, of the pyridoxine uptake process. These results demonstrate, for the first time, the involvement of a specialized carrier-mediated mechanism for pyridoxine uptake by intestinal epithelial cells. This system is pH dependent and amiloride sensitive and appears to be under the regulation of an intracellular PKA-mediated pathway.

Role of luminal anion and pH in distal tubule potassium secretion.

Potassium secretory flux (J(K)) by the distal nephron is regulated by systemic and luminal factors. In the present investigation, J(K) was measured with a double-barreled K(+) electrode during paired microperfusion of superficial segments of the rat distal nephron. We used control solutions (100 mM NaCl, pH 7.0) and experimental solutions in which Cl(-) had been replaced with a less permeant anion and/or pH had been increased to 8.0. J(K) increased when Cl(-) was replaced by either acetate ( approximately 37%), sulfate ( approximately 32%), or bicarbonate ( approximately 62%), and also when the pH of the control perfusate was increased ( approximately 26%). The majority (80%) of acetate-stimulated J(K) was Ba(2+) sensitive, but furosemide (1 mM) further reduced secretion ( approximately 10% of total), suggesting that K(+)-Cl(-) cotransport was operative. Progressive reduction in luminal Cl(-) concentration from 100 to 20 to 2 mM caused increments in J(K) that were abolished by inhibitors of K(+)-Cl(-) cortransport, i.e., furosemide and [(dihydroindenyl)oxy]alkanoic acid. Increasing the pH of the luminal perfusion fluid also increased J(K) even in the presence of Ba(2+), suggesting that this effect cannot be accounted for only by K(+) channel modulation of K(+) secretion in the distal nephron of the rat. Collectively, these data suggest a role for K(+)-Cl(-) cotransport in distal nephron K(+) secretion.