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.     

Acute experimental pancreatitis in rat induced by sodium taurocholic acid: objective quantification of pancreatic necrosis

Summary Retrograde injection of 5% sodium taurocholic acid (TA) in Wistar rat pancreatic duct is followed by acute pancreatitis, resulting in 100% mortality within 36 h. Biochemical determinations show raised levels of amylase in ascites and blood. Necrosis has been measured using seven morphometric characteristics of pathological changes that add precise information on the type and extension of the pancreatic lesion. The percentage of necrotic tissue (by area) seems to be the most objective parameter. Necrosis appears 6 h after TA infusion, being 5.77% in extent after 12h, 14.9% after 24 h and animals die with an area of 29.5% necrosis. This experimental model seems to one in which physiopathological and therapeutic trials on acute pancreatitis may be camed out.     

Evidence for a subgroup of essential hypertensives with non-suppressible excretion of aldosterone during sodium loading

Summary In patients with grade I and II essential hypertension studied during sodium loading (Na⁺ excretion above 175 meq·d^–1) we found a bimodal behaviour of aldosterone excretion and could distinguish two groups of patients: In the major part of essential hypertensives sodium loading led to a suppression of aldosterone excretion below 6 µg·d^–1, which is the highest control value during sodium loading, with an average of 2.7±1.4 (SD) µg·d^–1. Aldosterone excretion in a second group of patients was not suppressible below 6 µg·d^–1 despite forced sodium loading; it resulted in an average value of 10.0±3.0 (SD) µg·d^–1. During sodium deprivation or free sodium intake, aldosterone excretion in the first group of patients followed exactly the behaviour of normotensive controls, while in the second group of essential hypertensives the correlation of aldosterone excretion and log. Na excretion or log. Na⁺/K⁺ ratio in 24 h urine (r=–0.59) was far below the control value ofr=–0.87. Serum potassium concentration during sodium loading was significantly (p<0.001) lower (3.81±0.44 meq·l^–1) in the essential hypertensives with non-suppressible aldosterone excretion compared to those with suppressible aldosterone excretion (4.26±0.37 meq·l^–1). The blood pressure response to treatment with 200 mg spironolactone·d^–1 was better (p<0.05) in patients with non-suppressible aldosterone excretion compared to the essential hypertensives with normal aldosterone regulation. The plasma renin activity of both groups of patients was not significantly different, however, a tendency prevailed towards lower PRA-values in the patient group with non-suppressible aldosterone excretion during sodium loading.With the technical help of Mrs. R. Schendschilorz and Mrs. G. Suckau     

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)     

The effects of adenosine on transepithelial resistance and sodium uptake in the inner medullary collecting duct

It has been previously demonstrated that adenosine induces natriuresis when administered directly into the renal circulation of the rat. It was postulated that the mechanism was inhibition of tubule Na⁺ reabsorption. In the current study, the hypothesis was tested that adenosine inhibits ion reabsorption across the inner medullary collecting duct (IMCD), a tubule segment which is rich in adenosine receptors. IMCD epithelium from rat kidney was grown in primary culture as a confluent monolayer on Costar filters, allowing selective access to the basolateral and apical surfaces of the cells. Transepithelial resistance was taken as a measure of epithelial permeability and ion conductance. Na⁺ uptake was studied using ²²Na⁺ and used to determine the permeability of the epithelial monolayer specifically to Na⁺. Exposure of the basolateral aspect of the monolayer to adenosine (10⁻⁸–10⁻⁷ M) increased transepithelial resistance in a dose- and time-dependent manner; in parallel, adenosine (10⁻⁷–10⁻⁶ M) reduced apical Na⁺ uptake from 20±5 to 10±2 nmol/cm². 1,3-Dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX, 5×10⁻⁹ M), an adenosine antagonist with selectivity for the A₁ receptor, inhibited the rise in transepithelial resistance and the decrease in Na⁺ uptake following the addition of adenosine. The effects of adenosine on transepithelial resistance were reproduced with the A₁ receptor selective adenosine analogue N ⁶-cyclohexyladenosine (CHA, 10⁻⁸ –10⁻⁷ M) but not with the A₂ selective analogues, 5′-N-ethylcarboxamidoadenosine (NECA) or CGS 21680. CHA (10⁻⁷ M) inhibited apical Na⁺ uptake by 50%, an effect abolished by PACPX. The effects of adenosine on transepithelial resistance and Na⁺ uptake were inhibited, but only in part, by amiloride. These data suggest that adenosine inhibits ion movement, specifically apical Na⁺ uptake, across the IMCD epithelium and that this effect is mediated by A₁ receptors from the basolateral aspect of the cell. The results are consistent with the hypothesis that adenosine inhibits Na⁺ reabsorption across the IMCD.     

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.