Ultrastructural staining with sodium metaperiodate and sodium borohydride.

This article describes new ultrastructural staining methods for osmicated tissues based on the incubation of sections with sodium metaperiodate and sodium borohydride solutions before uranyl/lead staining. Sections incubated with sodium metaperiodate and sodium borohydride, treated with Triton X-100, and stained with ethanolic uranyl acetate/lead citrate showed a good contrast for the nucleolus and the interchromatin region, whereas the chromatin masses were bleached. Chromatin bleaching depended on the incubation with these oxidizing (metaperiodate) and reducing (borohydride) agents. Other factors that influenced the staining of the chromatin masses were the en bloc staining with uranyl acetate, the incubation of sections with Triton X-100, and the staining with aqueous or ethanolic uranyl acetate. The combination of these factors on sections treated with metaperiodate/borohydride provided a different appearance to the chromatin, from bleached to highly contrasted. Most cytoplasmic organelles showed a similar appearance with these procedures than with conventional uranyl/lead staining. However, when sections were incubated with metaperiodate/borohydride and Triton X-100 before uranyl/lead staining, the collagen fibers, and the glycocalix and zymogen granules of pancreatic acinar cells, appeared bleached. The possible combination of these methods with the immunolocalization of the amino acid taurine was also analyzed. (J Histochem Cytochem 50:11-19, 2002)     

Intracellular sodium activity and sodium transport inNecturus gallbladder epithelium

Summary Ion-sensitive glass microelectrodes, conventional microelectrodes and isotope flux measurements were employed inNecturus gallbladder epithelium to study intracellular sodium activity, [Na] _i , electrical parameters of epithelial cells, and properties of active sodium transport. Mean control values were: [Na] _i : 9.2 to 12.1mm; transepithelial potential difference, _ms : –1.5 mV (lumen negative); basolateral cell membrane potential, _es : –62 mV (cell interior negative); sodium conductance of the luminal cell membrane,g _Na: 12 mho cm^–2; active transcellular sodium flux, 88 to 101 pmol cm^–2 sec^–1 (estimated as instantaneous short-circuit current). Replacement of luminal Na by K led to a decrease of the intracellular sodium activity at a rate commensurate to the rate of active sodium extrusion across the basolateral cell membrane. Mucosal application of amphotericin B resulted in an increase of the luminal membrane conductance, a rise of intracellular sodium activity, and an increase of short-circuit current and unidirectional mucosa to serosa sodium flux. Conclusions: (i) sodium transport across the basolateral membrane can proceed against a steeper chemical potential difference at a higher rate than encountered under control conditions; (ii) the luminal Na-conductance is too low to accommodate sodium influx at the rate of active basolateral sodium extrusion, suggesting involvement of an electrically silent luminal transport mechanism; (iii) sodium entry across the luminal membrane is the rate-limiting step of transcellular sodium transport and active sodium extrusion across the basolateral cell membrane is not saturated under control conditions.     

Interaction of fatty acid sodium salts with sodium deoxycholate

Interaction of homologous fatty acids (C3-C18) with sodium deoxycholate was investigated. From NMR and ultrasonic results it was found that short chain homologues (up to C9) do not participate in the formation of mixed micelles with sodium deoxycholate. Fatty acid homologues with longer chains (starting with C9) form mixed micelles by "burying" hydrophobic chains in hydrophobic environment of a sodium deoxycholate micelle.     

Intracellular sodium regulates proteolytic activation of the epithelial sodium channel

Na(+) transport across epithelia is mediated in part by the epithelial Na(+) channel ENaC. Previous work indicates that Na(+) is an important regulator of ENaC, providing a negative feedback mechanism to maintain Na(+) homeostasis. ENaC is synthesized as an inactive precursor, which is activated by proteolytic cleavage of the extracellular domains of the alpha and gamma subunits. Here we found that Na(+) regulates ENaC in part by altering proteolytic activation of the channel. When the Na(+) concentration was low, we found that the majority of ENaC at the cell surface was in the cleaved/active state. As Na(+) increased, there was a dose-dependent decrease in ENaC cleavage and, hence, ENaC activity. This Na(+) effect was dependent on Na(+) permeation; cleavage was increased by the ENaC blocker amiloride and by a mutation that decreases ENaC activity (alpha(H69A)) and was reduced by a mutation that activates ENaC (beta(S520K)). Moreover, the Na(+) ionophore monensin reversed the effect of the inactivating mutation (alpha(H69A)) on ENaC cleavage, suggesting that intracellular Na(+) regulates cleavage. Na(+) did not alter activity of Nedd4-2, an E3 ubiquitin ligase that modulates ENaC cleavage, but Na(+) reduced ENaC cleavage by exogenous trypsin. Our findings support a model in which intracellular Na(+) regulates cleavage by altering accessibility of ENaC cleavage sites to proteases and provide a molecular explanation for the earlier observation that intracellular Na(+) inhibits Na(+) transport via ENaC (Na(+) feedback inhibition).     

The epithelial sodium channel and the control of sodium balance

Studies aiming at the elucidation of the genetic basis of rare monogenic forms of hypertension have identified mutations in genes coding for the epithelial sodium channel ENaC, for the mineralocorticoid receptor, or for enzymes crucial for the synthesis of aldosterone. These genetic studies clearly demonstrate the importance of the regulation of Na⁺ absorption in the aldosterone-sensitive distal nephron (ASDN), for the maintenance of the extracellular fluid volume and blood pressure.Recent studies aiming at a better understanding of the cellular and molecular basis of ENaC-mediated Na⁺ absorption in the distal part of nephron, have essentially focused on the regulation ENaC activity and on the aldosterone-signaling cascade. ENaC is a constitutively open channel, and factors controlling the number of active channels at the cell surface are likely to have profound effects on Na⁺ absorption in the ASDN, and in the amount of Na⁺ that is excreted in the final urine.A number of membrane-bound proteases, kinases, have recently been identified that increase ENaC activity at the cell surface in heterologous expressions systems. Ubiquitylation is a general process that regulates the stability of a variety of target proteins that include ENaC. Recently, deubiquitylating enzymes have been shown to increase ENaC activity in heterologous expressions systems.These regulatory mechanisms are likely to be nephron specific, since in vivo studies indicate that the adaptation of the renal excretion of Na⁺ in response to Na⁺ diet occurs predominantly in the early part (the connecting tubule) of the ASDN.An important work is presently done to determine in vivo the physiological relevance of these cellular and molecular mechanisms in regulation of ENaC activity. The contribution of the protease-dependent ENaC regulation in mediating Na⁺ absorption in the ASDN is still not clearly understood. The signaling pathway that involves ubiquitylation of ENaC does not seem to be absolutely required for the aldosterone-mediated control of ENaC. These in vivo physiological studies presently constitute a major challenge for our understanding of the regulation of ENaC to maintain the Na⁺ balance.     

Increased dietary sodium inhibits baroreflex-induced bradycardia during acute sodium loading

The present study investigated the effects of increased dietary sodium on the modification of cardiac baroreflex responses induced by acute sodium loading. Changes in blood pressure and heart rate during intravenous phenylephrine and nitroprusside administration were compared using a four-parameter sigmoid logistic function before and after a 30-min infusion of 0.6 or 1.0 M NaCl in conscious male Sprague-Dawley rats consuming only tap water (Tap) or isotonic saline (Iso) for 2-3 wk. In Tap animals, infusion of 1.0 M NaCl increased the baroreflex-induced heart rate minimum, reduced heart rate range, and increased the operating blood pressure. In contrast, infusion of 0.6 M NaCl in Tap rats reduced both heart rate minimum and maximum. However, infusion of 0.6 M NaCl in Iso animals produced responses similar to that shown in Tap rats infused with 1.0 M NaCl. In addition, the decreased heart rate minimum in Tap rats after infusion of 0.6 M NaCl was prevented by intravenous administration of a vasopressin V1-receptor antagonist. Furthermore, cardiac parasympathetic responses were similar in Tap and Iso rats before and after 0.6 M NaCl infusion. However, in animals receiving intravenous atropine, 0.6 M NaCl decreased heart rate minimum and maximum in Tap but did not alter the response parameters in Iso rats. These results demonstrate that the facilitation of cardiac baroreflex responses normally observed during moderate sodium loading is mediated by vasopressin and that increased dietary sodium ingestion reverses this facilitation by reducing sympathetic nervous system withdrawal.     

Cell sodium activity and sodium pump function in frog skin

Summary Cell Na activity,a _Na ^c , was measured in the short-circuited frog skin by simulaneous cell punctures from the apical surface with open-tip and Na-selective microelectrodes. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular conductance, with NaNO₃ Ringer on the apical surface. Under control conditionsa _Na ^c averaged 8±2mm (n=9,sd). Apical addition of amiloride (20 m) or Na replacement reduceda _Na ^c to 3mm in 6–15 min. Sequential decreases in apical [Na] induced parallel reductions ina _Na ^c and cell current,I _c . On restoring Na after several minutes of exposure to apical Na-free solutionI _c rose rapidly to a stable value whilea _Na ^c increased exponentially, with a time constant of 1.8±0.7 min (n=8). Analysis of the time course ofa _Na ^c indicates that the pump Na flux is linearly related toa _Na ^c in the range 2–12mm. These results indicate thata _Na ^c plays an important role in relating apical Na entry to basolateral active Na flux.     

Voltage-gated sodium channels

Epilepsy is a brain disorder characterized by seizures and convulsions. The basis of epilepsy is an increase in neuronal excitability that, in some cases, may be caused by functional defects in neuronal voltage gated sodium channels, Nav1.1 and Nav1.2. The effects of antiepileptic drugs (AEDs) as effective therapies for epilepsy have been characterized by extensive research. Most of the classic AEDs targeting Nav share a common mechanism of action by stabilizing the channel’s fast-inactivated state. In contrast, novel AEDs, such as lacosamide, stabilize the slow-inactivated state in neuronal Nav1.1 and Nav1.7 isoforms. This paper reviews the different mechanisms by which this stabilization occurs to determine new methods for treatment.     

Tetrodotoxin-resistant sodium channels

Summary 1. Tetrodotoxin (TTX) has been widely used as a chemical tool for blocking Na⁺ channels. However, reports are accumulating that some Na⁺ channels are resistant to TTX in various tissues and in different animal species. Studying the sensitivity of Na⁺ channels to TTX may provide us with an insight into the evolution of Na⁺ channels.2. Na⁺ channels present in TTX-carrying animals such as pufferfish and some types of shellfish, frogs, salamanders, octopuses, etc., are resistant to TTX.3. Denervation converts TTX-sensitive Na⁺ channels to TTX-resistant ones in skeletal muscle cells, i.e., reverting-back phenomenon. Also, undifferentiated skeletal muscle cells contain TTX-resistant Na⁺ channels. Cardiac muscle cells and some types of smooth muscle cells are considerably insensitive to TTX.4. TTX-resistant Na⁺ channels have been found in cell bodies of many peripheral nervous system (PNS) neurons in both immature and mature animals. However, TTX-resistant Na⁺ channels have been reported in only a few types of central nervous system (CNS). Axons of PNS and CNS neurons are sensitive to TTX. However, some glial cells have TTX-resistant Na⁺ channels.5. Properties of TTX-sensitive and TTX-resistant Na⁺ channels are different. Like Ca²⁺ channels, TTX-resistant Na⁺ channels can be blocked by inorganic (Co²⁺, Mn²⁺, Ni²⁺, Cd²⁺, Zn²⁺, La³⁺) and organic (D-600) Ca²⁺ channel blockers. Usually, TTX-resistant Na⁺ channels show smaller single-channel conductance, slower kinetics, and a more positive current-voltage relation than TTX-sensitive ones.6. Molecular aspects of the TTX-resistant Na⁺ channel have been described. The structure of the channel has been revealed, and changing its amino acid(s) alters the sensitivity of the Na⁺ channel to TTX.7. TTX-sensitive Na⁺ channels seem to be used preferentially in differentiated cells and in higher animals instead of TTX-resistant Na⁺ channels for rapid and effective processing of information.8. Possible evolution courses for Na⁺ and Ca²⁺ channels are discussed with regard to ontogenesis and phylogenesis.