Expression of Na+/HCO3- co-transporter proteins (NBCs) in rat and human skeletal muscle

AIM: Sodium/bicarbonate co-transport (NBC) has been suggested to have a role in muscle pH regulation. We investigated the presence of NBC proteins in rat and human muscle samples and the fibre type distribution of the identified NBCs. METHODS AND RESULTS: Western blotting of muscle homogenates and sarcolemmal membranes (sarcolemmal giant vesicles) were used to screen for the presence of NBCs. Immunohistochemistry was used for the subcellular localization. The functional test revealed that approximately half of the pH recovery in sarcolemmal vesicles produced from rat muscle is mediated by bicarbonate-dependent transport. This indicates that the NBCs are preserved in the vesicles. The western blotting experiments demonstrated the existence of at least two NBC proteins in skeletal muscle. One NBC protein (approximately 150 kDa) seems to be related to the kidney/pancreas/heart isoform NBC1, whereas the other protein (approximately 200 kDa) is related to the NBC4 isoform. The two NBC proteins represent the electrogenic isoforms named NBCe1 and NBCe2. Membrane fractionation and immunofluorescence techniques confirmed that the two NBCs are located in the sarcolemmal membrane as well as in some internal membranes, probably the T-tubules. The two NBCs localized in muscle have distinct fibre type distributions. CONCLUSIONS: Skeletal muscle possesses two variants of the sodium/bicarbonate co-transporter (NBC) isoforms, which have been called NBCe1 and NBCe2.     

Expression of the Na+/H+ exchanger isoform NHE1 in rat skeletal muscle and effect of training

The expression of the Na⁺/H⁺ exchanger isoform NHE1 was quantified in homogenates of various rat skeletal muscles by means of immunoblotting, and the effect of 3 weeks of treadmill training on NHE1 expression was determined in a red (oxidative) as well as a white (glycolytic)‐muscle preparation. The NHE1 antibodies recognized a glycosylated protein at 101–111 kDa. There was a positive correlation between the NHE1 expression in the muscle and percent type IIB fibres and percent type IID/X fibres, whereas the NHE1 expressions were negatively correlated to percent type I fibres and percent type I + IIA fibres. Thus the highest NHE1 expression was evident in the most glycolytic fibres. Treadmill training increased (P < 0.05) the NHE1 content by 29 and 36% in oxidative and glycolytic fibres, respectively, suggesting that training enhanced the NHE1 content of all muscle‐fibre types. Therefore training may improve the capacity for pH regulation in skeletal muscle.     

Expression and localization of Na-driven Cl-HCO(3)(-) exchanger (SLC4A8) in rodent CNS

The Na(+)-driven Cl-HCO(3) exchanger (NDCBE or SLC4A8) is a member of the solute carrier 4 (SLC4) family of HCO(3)(-) transporters, which includes products of 10 genes with similar sequences. Most SLC4 members play important roles in regulating intracellular pH (pH(i)). Physiological studies suggest that NDCBE is a major pH(i) regulator in at least hippocampal (HC) pyramidal neurons. We generated a polyclonal rabbit antibody directed against the first 18 residues of the cytoplasmic N terminus (Nt) of human NDCBE. By Western blotting, the antibody distinguishes NDCBE-as a purified Nt peptide or a full-length transporter (expressed in Xenopus oocytes)-from other Na(+)-coupled HCO(3)(-) transporters. By Western blotting, the antiserum recognizes an approximately 135-kDa band in several brain regions of adult mice: the cerebral cortex (CX), subcortex (SCX), cerebellum (CB), and HC. In CX, PNGase F treatment reduces the molecular weight to approximately 116 kDa. By immunocytochemistry, affinity-purified (AP) NDCBE antibody stains the plasma membrane of neuron cell bodies and processes of rat HC neurons in primary culture as well as freshly dissociated mouse HC neurons. The AP antibody does not detect substantial NDCBE levels in freshly dissociated HC astrocytes, or astrocytes in HC or CB sections. By immunohistochemistry, the AP antibody recognizes high levels of NDCBE in neurons of CX, HC (including pyramidal neurons in Cornu Ammonis (CA)1-3 and dentate gyrus), substantial nigra, medulla, cerebellum (especially Purkinje and granular cells), and the basolateral membrane of fetal choroid plexus. Thus, NDCBE is in a position to contribute substantially to pH(i) regulation in multiple CNS neurons.     

Expression of divalent metal transporter 1 (DMT1) isoforms in first trimester human placenta and embryonic tissues

BACKGROUND: Divalent metal transporter 1 (DMT1) is a transmembrane glycoprotein which mediates the proton-coupled transport of a variety of divalent metal ions. Two isoforms, which differ by the presence (DMT1-IRE) or absence (DMT1-nonIRE) of an iron-responsive element (IRE) in their 3' untranslated region, are implicated in apical iron transport and endosomal iron transport respectively. Although the expression pattern of DMT1 isoforms is tissue specific in adult, data regarding its expression in embryonic tissues are lacking. METHODS: Semiquantitative RT-PCR and immunohistochemistry were used to study the mRNA and protein expression of both DMT1 isoforms in embryonic tissues between 8 and 14 weeks gestational age. RESULTS: DMT1-IRE and DMT1-nonIRE expressions were ubiquitous in embryonic tissues examined. In the lung, statistically significant correlations were found between the levels of DMT1 isoform expression and gestational age. In the placenta, DMT1-IRE was the predominantly expressed isoform. Both isoform proteins were localized in embryonic epithelial cellular membrane. CONCLUSION: Both DMT1 isoforms are ubiquitously expressed in embryonic tissues in the first trimester. Predominant DMT1-IRE isoform expression in placenta suggests an iron-regulatory mechanism reminiscent of that in the adult duodenum. Epithelial distributions of both DMT1 isoforms are associated with the absorptive or excretory functions of the expressed tissues.     

Mutations and polymorphisms of the skeletal muscle α‐actin gene (ACTA1)

The ACTA1 gene encodes skeletal muscle α‐actin, which is the predominant actin isoform in the sarcomeric thin filaments of adult skeletal muscle, and essential, along with myosin, for muscle contraction. ACTA1 disease‐causing mutations were first described in 1999, when a total of 15 mutations were known. In this article we describe 177 different disease‐causing ACTA1 mutations, including 85 that have not been described before. ACTA1 mutations result in five overlapping congenital myopathies: nemaline myopathy; intranuclear rod myopathy; actin filament aggregate myopathy; congenital fiber type disproportion; and myopathy with core‐like areas. Mixtures of these histopathological phenotypes may be seen in a single biopsy from one patient. Irrespective of the histopathology, the disease is frequently clinically severe, with many patients dying within the first year of life. Most mutations are dominant and most patients have de novo mutations not present in the peripheral blood DNA of either parent. Only 10% of mutations are recessive and they are genetic or functional null mutations. To aid molecular diagnosis and establishing genotype–phenotype correlations, we have developed a locus‐specific database for ACTA1 variations ( http://waimr.uwa.edu.au ). Hum Mutat 30:1–11, 2009. © 2009 Wiley‐Liss, Inc.     

Role of physiological HCO3 –buffer on intracellular pH and histamine release in rat peritoneal mast cells

 The purpose of this study was to examine how intracellular pH (pH_i) regulation and histamine release are affected by HCO₃ ^–in rat peritoneal mast cells. The pH_i was measured using the pH-sensitive dye 2′, 7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). We observed a pH_i of 6.88±0.012 (n=24) in resting mast cells exposed to a HEPES buffer (pH 7.4), but a sustained drop of 0.21 pH units to 6.67±0.015 (n=23) when we exposed the mast cells to a HEPES/HCO₃ ^–buffer equilibrated at all time with 5% CO₂ (pH 7.4). This fall in pH_i is inhibited by the carbonic anhydrase inhibitor dichlorphenamide and is Na⁺-independent, indicating the involvement of Na⁺-independent Cl^–/HCO₃ ^–exchange activity. Furthermore removal of external Cl^–in the presence but not in the absence of HCO₃ ^–reversed the Cl^–/HCO₃ ^–exchange and induced an alkaline load. The recovery from this alkaline load was dependent on external Cl^–but independent of Na⁺. Both the alkalinization and the recovery were inhibited by the anion transport inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS). In addition, ³⁶Cl^–uptake measurements confirm the presence of a Cl^–/HCO₃ ^–exchanger. Histamine release stimulated by antigen and compound 48/80 was substantially reduced in the presence of HEPES/ HCO₃ ^–buffer (pH_o 7.4, pH_i 6.66). Histamine release was increased, however, when pH_i was clamped to 6.66 in HCO₃ ^–-free media (pH_o 6.9). We conclude that: (1) Na⁺-independent Cl^–/HCO₃ ^–exchange determines steady-state pH_i in rat peritoneal mast cells; and (2) the reduction in histamine release observed in the presence of HCO₃ ^–is not due to its effect on pH_i per se, but rather on other changes in ion transport. Received: 29 January 1998 / Received after revision and accepted: 3 April 1998     

Caffeine-induced depolarization in amphibian skeletal muscle fibres: role of Na+/Ca2+ exchange and K+ release

Caffeine (4 mM ) produces a depolarization of about 10 mV in frog muscle fibres (Leptodactylus ocellatus). The aim of this work was to study the mechanisms of this effect. An approximately threefold rise in membrane resistance [Cl⁻-free (SO₄^2–) medium] substantially increased, and both Na⁺-free medium and Ni²⁺ (5 mM ) reduced, the caffeine-induced depolarization. In voltage-clamped (–60 mV) short fibres from lumbricalis muscle of the toad (Buffo arenarum), caffeine generated an inward current of 4.13 ± 0.48 μA cm^–2. This caffeine-induced current was reduced by 60% in Na⁺-free medium, 44% in the presence of 5 mM amiloride and 48% by 5 mM Ni²⁺, suggesting that the activation of the Na⁺–Ca²⁺ exchanger in its forward mode may play a role in the observed electrical effects of the drug. Caffeine also produced a marked release of K⁺. Net K⁺ efflux increased from 3.5 ± 0.2 (control) to 22.1 ± 2.3 pmol s^–1 cm^–2 (caffeine). It is shown that in the presence of the drug, [K⁺] in the lumen of the T tubules may well increase to levels which could produce, in part, both the observed depolarization and the caffeine-induced current under voltage clamp conditions. The caffeine-induced K⁺ efflux was not reduced by 5 mM Ni²⁺. At a holding potential of 30 mV the caffeine-induced current was reversed (outward) and roughly halved by 5 mM Ni²⁺. The Ni²⁺-sensitive fraction of the caffeine-induced current, assumed to represent the Na⁺–Ca²⁺ exchanger current, had an estimated reversal potential close to 12 mV ([Na⁺]_o=115 mM ; [Ca²⁺]_o=1 mM ). In conclusion, the depolarizing effect of caffeine described here would be produced by two mechanisms: (a) an inward current generated by the activation of the Na⁺–Ca²⁺ exchanger in its forward mode, and (b) the rise of the external [K⁺] in restricted spaces like the T tubules.     

Lactate/proton co-transport in skeletal muscle: regulation and importance for pH homeostasis

In skeletal muscle, intracellular pH is more alkaline than would be predicted if H⁺ were passively distributed across the sarcolemma. Therefore, the passive influx of H⁺ must be counteracted by transport processes mediating H⁺ efflux. In resting skeletal muscle, these transport processes are Na⁺/H⁺ exchange and bicarbonate-dependent systems. During periods of high energy demand, skeletal muscle produces large amounts of lactic acid. The internal accumulation of lactic acid reduces pH, which may cause fatigue. It is therefore important for muscle cells to be able to regulate pH during and after activity. A part of the accumulated lactate and H⁺ is metabolized, but a considerable fraction is released from the cell. The efflux of H⁺ and lactate might be mediated by the lactate/proton co-transport system found in almost all cell types in the body. The role of lactate/proton co-transport in pH regulation has been studied both with intact cells and with sarcolemmal vesicles. In intact cells, inhibitors of lactate/proton transport have been shown to accelerate the development of fatigue, and to delay the recovery after activity. A comparison with vesicles has demonstrated that, at low pH, and with a high lactate concentration, the capacity for H⁺ removal is higher via the lactate/proton co-transport system than via the sum of the Na⁺/H⁺ exchange and bicarbonate-dependent exchange systems. Therefore, the carrier-mediated lactate/proton efflux is of major importance for pH regulation in connection with muscle activity. The lactate/proton transport system has been shown to undergo long-term changes depending on the level of physical activity. The capacity of the system was enhanced after intense training or chronic stimulation, and reduced after denervation. It is concluded that the lactate/proton transport system is of major importance for pH regulation in skeletal muscle, and that changes in the amount of transporters are one of the many adaptations to physical activity.     

Polarized expression of Na+/H+ exchange activities in clonal LLC-PK1 cells (Clone4 and PKE20)

We have analysed the mechanisms of Na⁺-dependent pH_i recovery from an acid load in LLC-PK₁/ Clone₄ and LLC-PK₁/PKE₂₀ cells by using the intracellular pH indicator 2,7-bis(carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester. By analysis using single-cell microspectrofluorometry, we obtained evidence for polarized expression of Na⁺/H⁺ exchange activities with different properties in apical and basolateral cell surfaces, respectively. In Clone₄ cells, Na⁺/H⁺ exchange activity is only visible on the basolateral cell surface; in PKE₂₀ cells, Na⁺/H⁺ exchange activities with equal capacities are present on both cell surfaces. In Clone₄ cells, the apparent K _m value for Na⁺ is around 10 mM; in PKE₂₀ cells it is around 20 mM and indistinguishable for the two cell poles. Ethylisopropylamiloride (EIPA) inhibition for all three activities measured in monolayer configuration is reduced by increasing Na⁺ concentration. Measured in the same cells, EIPA inhibition of transport of PKE₂₀ cells is weaker for apical Na⁺/H⁺ exchange as compared to basolateral activity. In Clone₄ and PKE₂₀ cells kept in suspension, Na⁺/H⁺ exchange activities with similar properties for the two cell lines are observed. However, Na⁺/H⁺ exchange activities in cells in suspension are different from either activity measured in monolayer configuration: affinity for Na⁺ is higher (PKE₂₀ cells) and inhibition by amiloride is weak and not influenced by increasing Na⁺ concentrations (PKE₂₀ and Clone₄ cells). It is concluded that PKE₂₀ cells contain different Na⁺/H⁺ exchange activities on the two cell surfaces; this cell line should be a useful model to study regulatory aspect of different Na⁺/H⁺ exchange functions (epithelial/housekeeping). Furthermore, measurements on cells in suspension do not reflect the properties of Na⁺/H⁺ exchange activities of cells in epithelial configuration.     

Networks Based on Poly(α‐methylene‐δ‐valerolactone‐co‐δ‐valerolactone): Crosslinking Through Free‐Radical Vinyl Copolymerization

Unsaturated polyesters are synthesized via ring‐opening copolymerization of α‐methylene‐δ‐valerolactone and δ‐valerolactone. These polyesters 4a–c are mixed with ethyl methacrylate (EMA), 2‐hydroxyethyl methacrylate (HEMA), and α‐methylene‐δ‐valerolactone (α‐MVL), respectively. Then, crosslinking is carried out by free radical polymerization initiated by an azo‐initiator. A second glass transition is found with incorporation of HEMA and α‐MVL. These findings indicate the formation of phase‐separated polyester blocks crosslinked with the poly(meth)‐acrylic‐segments, respectively poly(α‐methylene‐δ‐valerolactone) segments.

     

Skeletal muscle Na+/H+ exchange in rats: pH dependency and the effect of training

Skeletal muscle Na⁺/H⁺ exchange was studied using giant sarcolemmal vesicles obtained from rat hind limb muscle. Experiments with either the ²²Na tracer technique or with the Na⁺ sensitive fluorescent probe SBFI were conducted to determine the activity of the Na⁺/H⁺ exchanger, which was quantified from the amiloride or amiloride derivative 5-N-ethyl-N-propylamiloride (EIPA) sensitive Na⁺ influx. At a constant external pH of 7.4 the exchange system was close to half-activation at an internal pH of 7.2. A further activation was observed at lower internal pH values. The activity of the muscle Na⁺/H⁺ exchanger was elevated after 6 weeks of high-intensity treadmill training. In contrast, the activity of the system was unaffected by endurance training. The enhanced initial rate of amiloride-sensitive Na⁺/H⁺ exchange appears to be involved in the elevated in vivo (dynamical) buffer capacity reported for trained rats and human subjects, indicating that adaptive changes in the exchange system are of importance for pH regulation in association with high-intensity exercise.     

IgE and IL‐33−mediated triggering of human basophils inhibits TLR4−induced monocyte activation

Basophils are circulating granulocytes, best known as effector cells in allergic reactions. Recent studies in mice suggest that they might also participate in the suppression of chronic inflammation. The aim of this study was to assess the ability of purified human basophils to modulate monocyte responses upon IL‐33 and IgE triggering. Activation of human basophils with IL‐33 induced the production of IL‐4 and the release of histamine, and enhanced their IgE‐mediated activation. In addition, basophils triggered with IL‐33 and anti‐IgE significantly suppressed the LPS‐induced production of the proinflammatory cytokine TNF‐α and the upregulation of the costimulatory molecule CD80 by monocytes. These effects were mainly explained by the release of histamine, as they could be inhibited by the histamine receptor 2 antagonist ranitidine, with a smaller contribution of IL‐4. In contrast, basophil‐derived IL‐4 and histamine had opposing effects on the expression of the inhibitory Fc γ receptor IIb and the production of IL‐10 by monocytes. Our data show that basophils can influence monocyte activation and suggest a previously unrecognized role for human basophils in the modulation of monocyte‐mediated immune responses, through the balanced secretion of histamine and IL‐4.     

Effects of high‐intensity intermittent swimming on PGC‐1α protein expression in rat skeletal muscle

Aim: The aim of the present investigation was to elucidate the effects of exercise intensity on exercise‐induced expression of peroxisome proliferator‐activated receptor γ coactivator‐1α (PGC‐1α) protein in rat skeletal muscle. Methods: We measured PGC‐1α content in the skeletal muscles of male Sprague–Dawley rats (age: 5–6 weeks old; body weight: 150–170 g) after a single session of high‐intensity intermittent exercise (HIE) or low‐intensity prolonged swimming exercise (LIE). During HIE, the rats swam for fourteen 20‐s periods carrying a weight (14% of body weight), and the periods of swimming were separated by a 10‐s pause. LIE rats swam with no load for 6 h in two 3‐h sessions, separated by 45 min of rest. Results: After HIE, the PGC‐1α protein content in rat epitrochlearis muscle had increased by 126, 140 and 126% at 2, 6 and 18 h, respectively, compared with that of the age‐matched sedentary control rats’ muscle. Immediately, 6 and 18‐h after LIE, the PGC‐1α protein content in the muscle was significantly elevated by 84, 95 and 67% respectively. The PGC‐1α protein content observed 6 h after HIE tended to be higher than that observed after LIE. However, there was no statistically significant difference between the two values (P = 0.12). Conclusion: The present investigation suggests that irrespective of the intensity of the exercise, PGC‐1α protein content in rat skeletal muscle increases to a comparable level when stimuli induced by different protocols are saturated. Further, HIE is a potent stimulus for enhancing the expression of PGC‐1αprotein, which may induce mitochondrial biogenesis in exercise‐activated skeletal muscle.     

Molecular stressors underlying exercise training‐induced improvements in K+ regulation during exercise and Na+,K+‐ATPase adaptation in human skeletal muscle

Despite substantial progress made towards a better understanding of the importance of skeletal muscle K⁺ regulation for human physical function and its association with several disease states (eg type‐II diabetes and hypertension), the molecular basis underpinning adaptations in K⁺ regulation to various stimuli, including exercise training, remains inadequately explored in humans. In this review, the molecular mechanisms essential for enhancing skeletal muscle K⁺ regulation and its key determinants, including Na⁺,K⁺‐ATPase function and expression, by exercise training are examined. Special attention is paid to the following molecular stressors and signaling proteins: oxygenation, redox balance, hypoxia, reactive oxygen species, antioxidant function, Na⁺,K⁺, and Ca²⁺ concentrations, anaerobic ATP turnover, AMPK, lactate, and mRNA expression. On this basis, an update on the effects of different types of exercise training on K⁺ regulation in humans is provided, focusing on recent discoveries about the muscle fibre‐type‐dependent regulation of Na⁺,K⁺‐ATPase‐isoform expression. Furthermore, with special emphasis on blood‐flow‐restricted exercise as an exemplary model to modulate the key molecular mechanisms identified, it is discussed how training interventions may be designed to maximize improvements in K⁺ regulation in humans. The novel insights gained from this review may help us to better understand how exercise training and other strategies, such as pharmacological interventions, may be best designed to enhance K⁺ regulation and thus the physical function in humans.     

Evidence for Na/H exchange and Cl/HCO3 exchange in A10 vascular smooth muscle cells

In the present study we used the pH sensitive absorbance of 5(and6)-carboxy-4,5-dimethylfluorescein to investigate intracellular pH (pH_i) regulation in A10 vascular smooth muscle cells: (1) The steady state pH_i in A10 cells averaged 7.01±0.1 (mean±SEM,n=26) at an extracellular pH of 7.4 (28 mM HCO₃/5% CO₂). (2) Removal of extracellular sodium led to an intracellular acidification of 0.36±0.07 pH-units (mean±SEM,n=8). (3) pH_i-Recovery after an acute intracellular acid load (by means of NH₄Cl-prepulse) was reversibly blocked by 1 mM amiloride and was dependent on the presence of sodium. The velocity of pH_i recovery increased with increasing sodium concentrations with an apparentK _m for external sodium of about 30 mM and aV _max of about 0.35 pH units/min. These findings are compatible with a Na/H exchanger being responsible for pH_i recovery after an acid load. (4) Removal of extracellular chioride induced an intracellular alkalinization of 0.23±0.03 pH-units (mean±SEM,n=10). The alkalinization was dependent on the presence of extracellular bicarbonate (5) Removal of chloride during pH_i recovery from an alkaline load (imposed by acetate prepulse) stopped and reversed pH_i backregulation. Chloride removal had no effect in the absence of bicarbonate or in the presence of 10^–4 M DIDS, suggesting that the effects were mediated by a Cl/HCO₃ exchanger. In conclusion we have demonstrated evidence for a Na/H exchanger and a Cl/HCO₃ exchanger in A10 vascular smooth muscle cells.Abbreviations used CDMF 5(and6)-carboxy-4,5-dimethylfluorescein - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - NMDG N-methyl-d-glucamine; pH_i, intracellular pH - pH_o extracellular pH - Mops 3-[N-Morpholino]propanesulfonic acid - Hepes 2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid - Tris Tris(hydroxymethyl)-aminomethane - EDTA ethylenediamine-tetraacetic acid - EGTA ethyleneglycol-bis-(-amino-ethylether)N,N-tetraacetic acid