Functional expression and properties of the human skeletal muscle sodium channel

Full-length deoxyribonucleic acid, complementary (cDNA) constructs encoding the-subunit of the adult human skeletal muscle Na⁺ channel, hSkM1, were prepared. Functional expression was studied by electrophysiological recordings from cRNA-injectedXenopus oocytes and from transiently transfected tsA201 cells. The Na⁺ currents of hSkM1 had abnormally slow inactivation kinetics in oocytes, but relatively normal kinetics when expressed in the mammalian cell line. The inactivation kinetics of Na⁺ currents in oocytes, during a depolarization, were fitted by a weighted sum of two decaying exponentials. The time constant of the fast component was comparable to that of the single component observed in mammalian cells. The block of hSkM1 Na⁺ currents by the extracellular toxins tetrodotoxin (TTX) and -conotoxin (CTX) was measured. The IC₅₀ values were 25 nM (TTX) and 1.2 M (CTX) in oocytes. The potency of TTX is similar to that observed for the rat homolog rSkM1, but the potency of CTX is 22-fold lower in hSkM1, primarily due to a higher rate of toxin dissociation in hSkM1. Single-channel recordings were obtained from outside-out patches of oocytes expressing hSkM1. The single-channel conductance, 24.9 pS, is similar to that observed for rSkM1 expressed in oocytes.     

Effects of indomethacin on renal hemodynamics and on water and sodium excretion by the isolated dog kidney

Summary Blood-perfused isolated dog kidneys demonstrate steady increases in blood flow and in water and sodium excretion which could be attributed to the accumulation of renal prostaglandins in the perfusing blood. This hypothesis was tested by adding indomethacin, a potent inhibitor of prostaglandins synthesis, to the perfusing blood.Indomethacin completely prevented the vasodilation observed in control kidneys, without affecting glomerular filtration rate. Urine volume was not modified but sodium excretion was enhanced while the steady free water clearance increment observed in the control kidneys was depressed by indomethacin. The sum of sodium and free water clearances which, in the absence of antidiuretic hormone, constitutes an index of the part of the filtered load of solutes which escapes proximal tubular reabsorption, was not modified by indomethacin. Finally, indomethacin partially maintained the osmotic cortico-papillary gradient which was abolished after 2 hrs perfusion in control kidneys.These data suggest that prostaglandins accumulation in the blood is probably the major cause of the vasodilation taking place in isolated blood-perfused kidneys. This vasodilation does not account for decreased proximal reabsorption but partially explains the ADH-resistant diabetes insipidus developing in the isolated kidney. Moreover, indomethacin inhibits sodium reabsorption in the ascending limb of Henle's loop and increases water transport in the collecting duct.     

Effect of sodium ions on penicillin-induced epileptiform activity in vitro

Summary Intra- and extracellular recordings were obtained from the CA1 region of guinea pig hippocampal slices maintained in vitro. We studied the effect of reducing the extracellular sodium concentration on penicillin-induced epileptiform responses.In control experiments, Tris and choline were assayed as sodium substitutes. Choline was found unsuitable, since it induced repetitive firing in the absence of any convulsant agent. Replacement of 50% of the extracellular sodium ([Na⁺]_o) with Tris reduced the amplitude of the presynaptic fiber volley, the field EPSP, and the population spike. Intracellular studies showed that when [Na⁺]_o was lowered, action-potential amplitudes were reversibly depressed by an amount close to that predicted by the Nernst relation.Orthodromically elicited epileptiform discharges, induced by penicillin, were reduced in a low-sodium medium when constant stimulus currents were employed. If orthodromic stimulus strengths in normal and low-sodium states were equated on the basis of the field-EPSP amplitude, no significant diminution of the depolarizing-wave component of the epileptiform response was observed. These results suggest that a synaptic component underlies penicillin-induced epileptiform discharges.Supported by grants from the Norwegian Research Council for Science and the Humanities and by NIH grants NS 11535 and NS 15772     

Ionic dependence of electrical activity in small mesenteric arteries of guinea-pigs

The regulation of blood vessel diameter is under the control of the autonomic nervous system (as well as hormones and metabolites), sympathetic nerve stimulation evoking depolarizing post-synaptic potentials. Excitatory junction potentials (EJPs) were recorded from vascular smooth muscle cells of guinea-pig small mesenteric arteries (pressurized) following nerve stimulation. Repetitive stimulation (>5Hz) led to summation of EJPs, which evoked spikes and vasoconstriction. Replacing extracellular Na⁺ with choline (plus atropine) resulted in a decrease in EJP amplitude, but spike amplitude and maximum rate of rise (+V_max) were unaffected. Decreasing the extracellular Ca²⁺ concentration produced decreases in EJP amplitude and spike +V_max, while increasing extracellular Ca²⁺ resulted in increased EJP amplitude and spike +V_max. Verapamil and bepridil, agents that depress Ca²⁺ influx in vascular and visceral smooth muscle, depolarized the membrane and depressed EJPs and spikes at high concentrations (10^–5 M and 5×10^–6 M, respectively). The data indicate that EJPs are dependent on external Na⁺ and Ca²⁺ ions, and that spikes are dependent on Ca²⁺. Thus, neuromuscular transmission in this muscle is similar to that in non-vascular smooth muscles, such as intestinal muscle and vas deferens.Part of this work has been presented to the Biophysical Society (Zelcer and Sperelakis 1980) and to the American Physiological Society (Zelcer and Sperelakis 1981)     

Micropuncture studies on the filtration rate of single superficial and juxtamedullary glomeruli in the rat kidney

Summary Single nephron filtration rates of superficial and juxtamedullary nephrons were determined in high and low sodium rats. Single nephron GFR was calculated from TF/P inulin and tubular flow rate in superficial nephrons and single juxtamedullary GFR from corresponding data in long loops of Henle. In low sodium rats superficial nephron GFR was 23.5±6.4 (SD)×10^–6 ml/min×g KW, juxtamedullary nephron GFR was 58.2±13.6 and total kidney GFR (C _In) was 0.94±0.16 ml/min×g KW. Using these single nephron values, total kidney GFR and a total number of 30,000 glomeruli per kidney, the number of superficial and juxtamedullary glomeruli was calculated to be 23,267 and 6,733, respectively. During high sodium diet superficial nephron GFR increased to 38.1±11.3 and single juxtamedullary GFR decreased to 16.5±6.6, total kidney GFR increasing to 1.01±0.24. Calculation again revealed the same distribution of the two nephron types. End-proximal TF/P inulin in superficial nephrons was 2.36±0.36 in low sodium and 2.31±0.28 in high sodium rats. Loops of Henle TF/P inulin and intratubular flow rate were inversely related: the highest TF/P inulin values and lowest intratubular flow rates were found in the descending limb. These data quantify the distribution of superficial and juxtamedullary nephrons on a functional basis and suggest a mechanism by which the kidney adjusts sodium excretion by altering the contribution of each nephron type to total kidney GFR.Supported by the Deutsche Forschungsgemeinschaft and the U.S. Department of the Army, through its European Research Office.     

Kinetic mode switch of rat brain IIA Na channels in Xenopus oocytes excised macropatches

Na currents recorded from inside-out macropatches excised from Xenopus oocytes expressing the subunit of the rat brain Na channel IIA show at least two distinguishable components in their inactivation time course, with time constants differing about tenfold ( _h1 = approx. 150 s and _h2 = approx. 2 ms). In excised patches, the inactivation properties of Na currents changed with time, favoring the faster inactivation kinetics. Analysis of the fast and slow current kinetics shows that only the relative magnitudes of _h1 and _h2 components are altered without significant changes in the time constants of activation or inactivation. In addition, voltage dependence of both activation and steady-state inactivation of Na currents are shifted to more negative potentials in patches with predominantly fast inactivation, although reversal potentials and valences remained unaltered. We conclude that the two inactivation modes discerned in this study are conferred by two states of Na channel the interconversion of which are regulated by an as yet unknown mechanism that seems to involve cytosolic factors.     

The mechanisms of fast renal compensation

Summary The changes in blood concentration which result in the adjustment of excretion when renal functioning mass is acutely reduced have been investigated by means of paired experiments on isolated dog kidneys. One kidney was perfused with undiluted blood; the other kidney was perfused with blood supplemented with an amount of water and solutes corresponding to the amount retained after the suppression of the contralateral kidney in situ; the response was evaluated from the difference in excretion between the two organs. The results may be summarized as follows: a) the adjustment of the excretion of water, sodium, potassium and urea results from small changes in blood concentration (haematocrit, plasma proteins and solutes), in the absence of specific stimuli of extrarenal origin; b) increased urea concentration is not the major determinant of the readjustment of sodium and water excretion; c) the response is potentiated by high arterial blood pressure; d) if the load of water exceeds the load of sodium, this ion is retained by the kidney even in the presence of an osmotic load of urea; e) the changes in the blood concentrations do not provide an adequate adjustment of the excretion of phosphate; f) increased excretion per nephron results from decreased fractional reabsorption without significant change in glomerular filtration rate.     

Sodium regulation of agonist and antagonist binding to β-adrenoceptors in intact and Ns-deficient membranes

Summary Agonist binding to various hormone receptors mediating adenylate cyclase inhibition is decreased by sodium ions. We studied the influence of Na⁺ on agonist and antagonist binding to -adrenoceptors in membrane preparations of guinea pig lung, S49 lymphoma wild-type cells (WT) and their N_s-deficient cyc^– variants by measuring binding of the antagonist, [¹²⁵I]iodocyanopindolol ([¹²⁵I]CYP). At 37° C, sodium decreased the receptor affinity for the agonist, isoproterenol, in all three membrane preparations. In lung and WT membranes, Na⁺ steepened the shallow agonist competition curves in a manner similar to and synergistic with guanine nucleotides. When binding was performend at 4° C, sodium regulation but not guanine nucleotide regulation of agonist binding was preserved. At the low temperature, [¹²⁵I]CYP affinity was reduced, and sodium increased [¹²⁵I]CYP binding in both N_s-containing and N_s-deficient membranes by increasing the antagonist affinity without significant change in total receptor number. Compared to Na⁺, Li⁺ and K⁺ were much less potent and efficient in decreasing agonist and increasing antagonist binding. Na⁺ and Mg²⁺ had opposite effects on agonist binding in the N_s-containing lung and WT membranes but not in the N_s-deficient cyc^– membranes. The data indicate that sodium not only regulates binding of inhibitory hormone receptors but also agonist and antagonist binding to the adenylate cyclase stimulatory -adrenoceptor. The finding that sodium regulation of -adrenoceptor binding is also observed in the N_{s 2} cyc^– membranes, furthermore, indicates that the target of sodium is not the -subunit of N_s but possibly a component common to both types of receptor systems regulating adenylate cyclase activity.     

含有十二烷基磺酸钠的生物制剂的内毒素检测不能采用鲎试验

重组人ＩＬ－２在大肠杆菌表达时形成包含体，需经过变性和复性才具有生物学活性，在分离提取过程中常加一定量的ＳＤＳ，最低浓度每毫升不小于０．２５ｍｇ；低于此浓度会发生沉淀。鲎试验（ＬＡＬ试验）是一种灵敏度很高的检查生物制剂中致热源的常规方法。我们发现，当样本中ＳＤＳ的浓度＞０．０２５ｍｇ／ｍｌ时，可抑制尝试制的酶反应，使阳性结果转变为阴性。据此，我们建议，凡生物制剂中含有ＳＤＳ或其他酶抑制剂的产品，以尝试验来检查内毒素含量是没有意义的。