Expression of rat ileal Na+-sulphate cotransport in Xenopus laevis oocytes: functional characterization

Small-intestinal sulphate absorption is a Na⁺-dependent process having its highest rate in the ileum; it involves brush-border membrane Na⁺-sulphate cotransport. Injection of rat ileal mRNA into Xenopus laevis oocytes induced Na⁺-dependent sulphate uptake in a dose-dependent manner, with no apparent effect on Na⁺-independent sulphate uptake. For mRNA-induced transport, the apparent K _m value for sulphate interaction was 0.6±0.2 mM and that for sodium interaction was 25±2 mM (Hill coefficient: 2.3±0.3). mRNA-induced transport, was inhibited by thiosulphate, but not by phosphate or 4,4,′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS). Using a rat renal Na⁺-sulphate cotransporter cDNA as a probe [NaSi-1; Markovich et al. (1993) Proc Natl Acad Sci USA 90:8073–8077], the highest hybridization signals (2.3 kb and 2.9 kb) were obtained in size fractions showing the highest expression of Na⁺-dependent sulphate transport in oocytes. Hybrid depletion experiments using antisense oligonucleotides (from the NaSi-1 cDNA sequence), provided further evidence that rat small-intestinal (ileal) Na⁺-sulphate cotransport is closely related to rat proximal-tubular brushborder membrane Na⁺-sulphate cotransport.     

Modulation of Na+,K+‐ATPase activity is of importance for RVD

Aim: This study was performed to examine the role of Na⁺,K⁺‐ATPase activity for the adaptive response to cell swelling induced by hypoosmoticity, i.e. the regulatory volume decrease (RVD). Methods: The studies were performed on COS‐7 cells transfected with rat Na⁺,K⁺‐ATPase. To study changes in cell volume, cells were loaded with the fluorescent dye calcein and the intensity of the dye, following exposure to a hypoosmotic medium, was recorded with confocal microscopy. Results: Ouabain‐mediated inhibition of Na⁺,K⁺‐ATPase resulted in a dose dependent decrease in the rate of RVD. Total ⁸⁶Rb⁺ uptake as well as ouabain dependent ⁸⁶Rb⁺ uptake, used as an index of Na⁺,K⁺‐ATPase dependent K⁺ uptake, was significantly increased during the first 2 min following exposure to hypoosmoticity. Since protein kinase C (PKC) plays an important role in the modulation of RVD, a study was carried out on COS‐7 cells expressing rat Na⁺,K⁺‐ATPase, where Ser23 in the catalytic α1 subunit of rat Na⁺,K⁺‐ATPase had been mutated to Ala (S23A), abolishing a known PKC phosphorylation site. Cells expressing S23A rat Na⁺,K⁺‐ATPase exhibited a significantly lower rate of RVD and showed no increase in ⁸⁶Rb⁺ uptake during RVD. Conclusion: Taken together, these results suggest that a PKC‐mediated transient increase in Na⁺,K⁺‐ATPase activity plays an important role in RVD.     

Distribution of sodium-potassium ATPase in the rat testis and epididymis

Sodium-potassium ATPase (Na⁺ K⁺-ATPase) is a ubiquitous plasma membrane enzyme which uses the hydrolysis of ATP to regulate cellular Na⁺ and K⁺ levels and fluid volume. This ion pumping action is also thought to be involved in fluid movement across certain epithelia. There are several different genes for this enzyme, some of which are tissue specific. Using an antibody specific for the catalytic subunit of canine kidney Na⁺ K⁺-ATPase, we have localized immunoreactivity in the seminiferous and epididymal epithelium of rats of various ages. There was no specific staining of 10-day-old rat testis. Faint staining was detected at 13 days and appeared to be associated with the borders of Sertoli cells. At 16 days prominent apical and lateral staining but no basal staining of Sertoli cell membranes was observed. This type of distribution continued until spermatids were present in the epithelium. In the adult rat testis, specific staining was detected in Sertoli cell crypts associated with elongating spermatids, and on the apical and lateral Sertoli cell membrane. In some instances immunoreactivity was concentrated at presumed sites of junctional specializations. In the excurrent ducts of immature and mature rats, Na⁺ K⁺-ATPase staining was heavy in the efferent ducts and somewhat lighter in the epididymis. In all regions, the staining was basolateral although there were variations in intensity among the different parts of the epididymis. These results show (1) that rat testis and epididymal Na⁺ K⁺-ATPase share some immunological determinants with the canine enzyme; (2) that the epididymal enzyme is located in the conventional basolateral position; and (3) that the distribution of Sertoli cell Na⁺ K⁺-ATPase is probably apical and lateral rather than basal.     

On the functional interaction between nicotinic acetylcholine receptor and Na+,K+-ATPase

Previous studies have shown that nanomolar acetylcholine (ACh) produces a 2 to 4-mV hyperpolarization of skeletal muscle fibers putatively due to Na⁺,K⁺-ATPase activation. The present study elucidates the involvement of the nicotinic ACh receptor (nAChR) and of Na⁺,K⁺-ATPase isoform(s) in ACh-induced hyperpolarization of rat diaphragm muscle fibers. A variety of ligands of specific binding sites of nAChR and Na⁺,K⁺-ATPase were used. Dose–response curves for ouabain, a specific Na⁺,K⁺-ATPase inhibitor, were obtained to ascertain which Na⁺,K⁺-ATPase isoform(s) is involved. The ACh dose–response relationship for the hyperpolarization was also determined. The functional relationship between these two proteins was also studied in a less complex system, a membrane preparation from Torpedo electric organ. The possibility of a direct ACh effect on Na⁺,K⁺-ATPase was studied in purified lamb kidney Na⁺,K⁺-ATPase and in rat red blood cells, systems where no nAChR is present. The results indicate that binding of nAChR agonists to their specific sites results in modulation of ouabain-sensitive (most probably α2) isoform of Na⁺,K⁺-ATPase, leading to muscle membrane hyperpolarization. In the Torpedo preparation, ouabain modulates dansyl-C6-choline binding to nAChR, and vice versa. These results provide the first evidence of a functional interaction between nAChR and Na⁺,K⁺-ATPase. Possible interaction mechanisms are discussed.     

Sodium-dependent regulation of sodium,potassium-adenosine-tri-phosphatase (Na+, K+-ATPase) activity in medullary thick ascending limb of Henle segments. Effect of cyclic-adenosine-monophosphate guanosine-nucleotide-binding-protein activity and arginine vasopressin

This study examine the regulation Na⁺, K⁺-ATPase activity in the medullary thick ascending limb of Henle Na⁺, K⁺-ATPase activity was determined in medullary thick ascending limb of Henle (mtal) segments dissected from rat kidneys. The sodium concentration in the medium (Na,) was 20 or 70 mM. Since the segments were permeabilized, intracellular Na⁺ (Na,) was assumed to be the same as Na₂. Dibuturyl cyclic adenosine monophosphate (dbcAMP) and forskolin inhibited Na⁺, K⁺-ATPase activity independently of Na_m. Arginine vasopressin (AVP) receptors coupled to adenylate cyclase have been identified in the medullary thick ascending limb of Henle. At Na_m= 20 mMAVP caused a dose-dependent inhibition of Na⁺, K⁺-ATPase activity with a maximal effect (49%) at 10^-8 m. This inhibition was abolished in the presence of the adenylate cyclase inhibitor 2,5-dideoxyadenosine (2, 5-DDA). AVP had no effect on Na⁺, K⁺-ATPase activity in the mTAL at Na_m= 70 mM. The guanosine-diphosphate analogue GDPβS inhibited Na⁺, K⁺-ATPase activity at Na_m= 70 mM but not at Na_m= 20 mM. We conclude that increased cyclic adenosine monophosphate (CAMP) levels inhibit Na⁺, K⁺, ATPase activity in mTAL. AVP can, depending on Na₂ produce this effect by adenylate cyclase activation. The guanonine nucleotide binding protein G-protein might be the site of Na⁺-dependence.     

The regulation of the Na+,K+ pump in contracting skeletal muscle

Increased passive Na⁺,K⁺ fluxes necessitate an efficient activation of the Na⁺,K⁺ pump in working muscles to limit the rundown of the Na⁺,K⁺ chemical gradients and ensuing loss of excitability. Several studies have demonstrated an increase in Na⁺,K⁺-pump rate in working muscles, and in electrically stimulated muscles up to a 22-fold increase in active Na⁺,K⁺ transport has been observed. Excitation-induced increase in intracellular Na⁺ is believed to be the primary stimulus for Na⁺,K⁺ pumping in a contracting muscle. In muscles recovering from electrical stimulation, however, the activity of the pump may stay elevated even after intracellular Na⁺ has been reduced to below the resting level. Moreover, in rat soleus muscles 10-s stimulation at 60 Hz induced a 5-fold increase in the activity of the Na⁺,K⁺ pump although mean intracellular [Na⁺] was unchanged. These findings strongly suggest that a substantial part of the excitation-induced increase in Na⁺,K⁺-pump activity is caused by mechanisms other than increased intracellular [Na⁺]. The mechanism behind this activation is not clear, but may involve a change in the affinity of the Na⁺,K⁺ pump for intracellular Na⁺. In addition to intracellular [Na⁺], the Na⁺,K⁺ pump may be stimulated in contracting muscles by other factors such as catecholamines, calcitonin gene-related peptide (CGRP), free fatty acids and cytoskeletal links. Together, this activation may form a feed forward mechanism protecting muscles from loss of excitability during periods of contraction by increasing Na⁺,K⁺-pump activity prior to erosion of the Na⁺,K⁺ chemical gradients. During exercise of high intensity, however, intracellular [Na⁺] increases substantially constituting an additional stimulus for the pump.     

An early outward transient K+ current that depends on a preceding Na+ current and is enhanced by insulin

A whole-cell early transient outward current occurs in rat myoballs if and only if there is an immediatly preceding current of large amplitude through the voltage-gated, tetrodotoxin-inhibitable Na⁺ channel. This early outward transient is a K⁺ current, designated I _K(Na⁺). Under the conditions in which I _K(Na⁺) appears, simultaneous measurement of voltage and current, under voltage clamp, demonstrates that there is transient voltage escape to depolarized levels, peaking at about the time of peak inward Na⁺ current arid resembling an action potential. I _K(Na⁺) was never seen in the absence of this breach of the voltage clamp, suggesting that I _K(Na⁺) might be an artefact due to transient depolarization from the clamp. However, when the voltage escape was mimicked by voltage commands under conditions in which the Na⁺ channel was not activated, there was no I _K(Na). Insulin increased or produced I _K(Na⁺) even though insulin had no effect on I _Na or on the delayed rectifier K⁺ current or on the escape from voltage clamp. It is concluded that there is a population of rat myoballs in which there is an early outward K⁺ current that requires an immediately preceding current through the voltagegated tetrodotoxin-inhibitable Na⁺ channel and is enhanced by insulin.     

Occurrence and properties of a (Na+−K+)-activated ATPase in the mucosa of the rat intestine

Summary The existence of an ouabain-sensitive (Na⁺–K⁺)-activated ATPase system has been demonstrated in the total intestine of the rat. The (Na⁺–K⁺)-ATPase activity was about 10–15% of the total ATPase in 4 equal parts of the small intestine; in the colon about 35% of the total ATPase was (Na⁺–K⁺)-activated ATPase. The highest (Na⁺–K⁺)-ATPase activity has been observed in the first and second part of the small intestine, while in the colon the activity was 2–4 times higher than in the ileum.The (Na⁺–K⁺)-ATPase of rat colon required both Na⁺ (K _m=8.3 mmoles/l) and K⁺ (K _m=0.6 mmoles/l). Maximal activation of the (Na⁺–K⁺)-ATPase system required 2 mM Mg²⁺ at an ATP concentration of 2 mM. The pH optimum for (Na⁺–K⁺)-ATPase of rat colon was 7.5, while the Mg²⁺-activated ATPase activity had a pH optimum of 8.6. The (Na⁺–K⁺)-ATPase was inhibited by ouabain (pI ₅₀=3.6).The relation between the differences in (Na⁺–K⁺)-ATPase activity and Na⁺-absorption on different parts of the intestine is discussed.     

Distribution of (Na+ + K+)ATPase and sodium channels in skeletal muscle and electroplax

Summary The distributions of (Na⁺ + K⁺)ATPase and sodium channels in skeletal muscle fibres and electrocytes were determined by immunofluorescent and immunoelectron microscopic techniques using antibodies against rat and eel (Na⁺ + K⁺)ATPase and the eel electric organ sodium channel. The extrajunctional sarcolemma of skeletal muscle was uniformly stained by polyclonal antibodies against (Na⁺ + K⁺)ATPase and the sodium channel. The T-tubule system of skeletal muscle was also labelled heavily for both (Na⁺ + K⁺)ATPase and the sodium channel. The terminal cisternae of the sarcoplasmic reticulum was stained for (Na⁺ + K⁺)ATPase but not sodium channels. At the motor endplate, (Na⁺ + K⁺)ATPase-like immunoreactivity was present along the plasmalemma of motor nerve terminals but not along the postsynaptic junctional sarcolemma. Paradoxically, a monoclonal antibody that binds to the form of the catalytic subunit of (Na⁺ + K⁺)ATPase from rat hepatocytes and renal tubule cells did not label the enzyme in rat skeletal muscle. In electrocytes, (Na⁺ + K⁺)ATPase-like irnmunoreactivity was concentrated primarily along the plasmalemma and calveolae of the non-innervated face. In contrast, sodium channel-like immunoreactivity was concentrated along the plasmalemma of the innervated face except in the clefts of the postsynaptic membrane. Thus, we conclude that at endplates both the (Na⁺ + K⁺)ATPase of rat skeletal muscle and sodium channels of eel electrocytes are not concentrated in the juxtaneuronal postsynaptic membrane. We also interpret the failure of the monoclonal anti- (Na⁺ + K⁺)ATPase antibodies to bind to the enzyme in muscle to indicate that the catalytic subunit of skeletal muscle (Na⁺ + K⁺)ATPase displays different epitopes than does the a subunit of kidney and liver.     

Na+ and pH dependence of proline and beta-alanine absorption in rat small intestine

Aims: Early characterization of intestinal absorption of imino acids in mammals has demonstrated the existence of a Na⁺‐dependent, Cl^?‐independent transport system in rat small intestine, which is the only carrier for β‐alanine. Based on the substrate selectivity, it was proposed that the Proton Amino Acid Transporter 1 (PAT1) could be the same as this imino acid carrier. The present study characterizes the pH and Na⁺ dependence of proline and β‐alanine uptake in rat small intestine. Methods: Intestinal uptake of radiolabelled l ‐proline or β‐alanine was measured in brush border membrane vesicles and everted intestinal rings, in the presence and absence of Na⁺ and at different pH values. Results: The existence of an inwardly directed H⁺ gradient in the absence of Na⁺ enhanced the initial entry of proline and β‐alanine in brush border membrane vesicles, that reached a transient overshoot with maximal value around 30 s. In the absence of pH gradient, no overshoot was shown. In entire tissue, there was an increase of proline and β‐alanine uptake at acidic pH that was higher in the presence of Na⁺ than in its absence. This ion dependence and pH effect of the amino acids uptake also increased with the incubation period. Substrate inhibition studies confirmed that intestinal proline absorption in rat occurs mainly by system B and PAT1‐like transporter. Conclusions: There is a Na⁺‐independent, H⁺‐dependent transporter of amino acids at the apical membrane of the rat enterocytes.     

Intracellular sodium modulates the state of protein kinase C phosphorylation of rat proximal tubule Na+,K+‐ATPase

The natriuretic hormone dopamine and the antinatriuretic hormone noradrenaline, acting on α‐adrenergic receptors, have been shown to bidirectionally modulate the activity of renal tubular Na⁺,K⁺‐adenosine triphosphate (ATPase). Here we have examined whether intracellular sodium concentration influences the effects of these bidirectional forces on the state of phosphorylation of Na⁺,K⁺‐ATPase. Proximal tubules dissected from rat kidney were incubated with dopamine or the α‐adrenergic agonist, oxymetazoline, and transiently permeabilized in a medium where sodium concentration ranged between 5 and 70 mM . The variations of sodium concentration in the medium had a proportional effect on intracellular sodium. Dopamine and protein kinase C (PKC) phosphorylate the catalytic subunit of rat Na⁺,K⁺‐ATPase on the Ser23 residue. The level of PKC induced Na⁺,K⁺‐ATPase phosphorylation was determined using an antibody that only recognizes Na⁺,K⁺‐ATPase, which is not phosphorylated on its PKC site. Under basal conditions Na⁺,K⁺‐ATPase was predominantly in its phosphorylated state. When intracellular sodium was increased, Na⁺,K⁺‐ATPase was predominantly in its dephosphorylated state. Phosphorylation of Na⁺,K⁺‐ATPase by dopamine was most pronounced when intracellular sodium was high, and dephosphorylation by oxymetazoline was most pronounced when intracellular sodium was low. The oxymetazoline effect was mimicked by the calcium ionophore A23187. An inhibitor of the calcium‐dependent protein phosphatase, calcineurin, increased the state of Na⁺,K⁺‐ATPase phosphorylation. The results imply that phosphorylation of renal Na⁺,K⁺‐ATPase activity is modulated by the level of intracellular sodium and that this effect involves PKC and calcium signalling pathways. The findings may have implication for the regulation of salt excretion and sodium homeostasis.     

Expression of a renal Na+-nucleoside cotransport system (N2) in Xenopus laevis oocytes

Xenopus laevis oocytes have been used for the expression of a renal, pyrimidine-selective, Na⁺-nucleoside cotransporter (N2). As compared to its uptake in water-injected oocytes, Na⁺-dependent thymidine uptake was enhanced in a time- and dose- dependent manner in oocytes injected with rat renal cortex total poly(A)⁺ RNA. An increased uptake was also observed after injection of size fractionated rat renal cortex poly(A)⁺ RNA (2–3 kb). Consistent with the selectivity of the N2 nucleoside transporter, cytidine significantly inhibited Na⁺-dependent thymidine uptake in oocytes injected with total poly(A)⁺ RNA whereas guanosine and formycin B did not. Na⁺-dependent thymidine uptake was also enhanced in oocytes injected with size fractionated human renal cortex poly(A)⁺ RNA (2–3 kb). The above data demonstrate functional expression of renal cortex, Na⁺-nucleoside cotransporters in Xenopus laevis oocytes.     

The mechanism of action of harmaline on renal solute transport

Summary The effect of the hallucinogenic drug harmaline was tested on rat kidney proximal tubular solute and water transport, using in vivo micropuncture and electrophysiological techniques as well as in vitro biochemical techniques. During peritubular application harmaline (5 mmol/l) was found to block net tubular volume absorption reversibly (by 85%) through inhibition of active Na⁺ transport and possibly active HCO ₃ ^– transport. The inhibition was accompanied by a rapid strong depolarization of the tubular cell membranes. As a biochemical equivalent harmaline inhibited the Na⁺–K⁺-ATPase and the Mg²⁺-ATPase of peritubular cell membrane fractions as well as the HCO ₃ ^– -stimulated ATPase of a brush border membrane fraction with similar kinetics. By studying glucose tracer efflux and by measuring cell membrane potential and conductance changes in response to glucose perfusions, no evidence for a direct effect of harmaline on Na⁺-glucose (or amino acid) cotransport mechanisms in the brush border could be obtained. The data suggest that harmaline does not specifically compete with Na⁺ for transport sites. Neither are the cotransport systems in the brush border membrane specifically inhibited, nor could the inhibition of the Na⁺ pump in the peritubular cell membrane simply result from a competition between harmaline and Na⁺.     

Ionic mechanisms of alpha-autoinhibition of [3H]noradrenaline secretion in guinea-pig vas deferens: possible role of extracellular sodium

Guinea-pig isolated vas deferens, in which the neuronal transmitter stores were labelled by preincubation with [³H](?)-noradrenaline was used to study ionic mechanisms of α-autoinhibition of the secretion of [³H]noradrenaline evoked by transmural electrical stimulation or by depolarizing concentrations of K⁺. Replacement of Na⁺ in the medium markedly enhanced K⁺-evoked secretion, and thus probably improved depolarization-secretion coupling. It had a dual effect on the tetrodotoxinsensitive electrically-evoked secretion: at sufficiently low Na⁺ levels the secretory response was severely depressed, presumably because of failure of impulses to invade the varicosities. At intermediate Na⁺ concentrations the secretory response was greater than at normal Na⁺ levels. It is concluded that extracellular Na⁺ depresses depolarization-secretion coupling in invaded varicosities. The depressing effect of extracellular Na⁺ did not appear to be due to lowering of the affinity of the secretory mechanism for external Ca²⁺. The inhibitory effect of exogenous or endogenous noradrenaline, or of prostaglandin E₂, on electrically or K⁺-evoked secretion, was reduced or abolished at low concentrations of Na⁺.It is suggested that the α-adrenoceptor mediated inhibition of depolarization-secretion coupling might involve the same mechanism by which extracellular sodium ions inhibit the secretion of noradrenaline.     

Enzyme histochemical localization of Na+,K+‐ATPase and NADH‐DE in the developing rat parotid gland

Information on ductal differentiation in the developing rat parotid gland is sparse. Striated and excretory ducts are rich in a number of enzymes related to ion movement. The objective of this investigation was to delineate histochemically the chronology of two of these, ouabain‐sensitive Na⁺,K⁺‐ATPase and NADH‐DE, in the developing rat parotid gland. Parotid glands were excised from rats at representative ages from 20 days in utero to 42 days. Enzyme histochemistry was performed on air‐dried frozen sections. For Na⁺,K⁺‐ATPase, some sections also were fixed in phosphate‐buffered formalin. Ouabain blocked Na⁺,K⁺‐ATPase activity, and neither enzyme reacted without substrate. Weak Na⁺,K⁺‐ATPase reactions were initially seen in unfixed sections at 1 day, and increased steadily to the adult pattern of strong (concentrated basolaterally) in striated ducts and excretory ducts, respectively, and weak to modest (diffuse) in acini and intercalated ducts at 28 days. In fixed sections, localization was sharper but the reaction was somewhat reduced. NADH‐DE was modest in terminal buds and ducts before birth, then progressively changed to the adult pattern of weak in acini and intercalated ducts and strong (concentrated basally and luminally) in striated and excretory ducts at 28 days. As demonstrated by enzyme histochemistry of Na⁺,K⁺‐ATPase and NADH‐DE, differentiation of rat parotid striated ducts and excretory ducts occurs mainly between birth and 28 days. Anat Rec 256:72–77, 1999. Published 1999 Wiley‐Liss, Inc.     

Influence of bile acids on the (Na+−K+)-activated- and Mg2+-activated ATPase of rat colon

Summary The influence of various bile acids on the (Na⁺−K⁺)-ATPase and Mg²⁺-ATPase activity of rat colon is described. At a concentration of 0.6 mmol/l C and TC did not inhibit the (Na⁺−K⁺)-ATPase activity in contrast to GC. The taurine derivates TC, TCDC and TDC did not influence or even enhanced the (Na⁺−K⁺)-ATPase activity. All bile acids except C, TC and CDC depressed the Mg²⁺-ATPase activity. At higher concentrations only C and TC did not influence the (Na⁺−K⁺)-ATPase activity. C, GC and TC at 2.5 mmol/l decreased the (Na⁺−K⁺)-activated phosphatase with ATP as substrate. All other substrates tested did not influence the enzymic activity significantly. The results indicate that bile acids can inhibit the Na⁺-absorbing system in rat colon. Hence this inhibition can cause diarrhea.     

Different functional states of tetrodotoxin sensitive and tetrodotoxin resistant Na+ channels occur during the in vitro development of rat skeletal muscle

There are three stages of differentiation of voltage dependent Na⁺ channels during the in vitro development of rat skeletal muscle.

1. Myoblasts which are less than 60 h old in culture have Na⁺ channels which normally do not give rise to action potentials but do so after treatment of the cells with very low concentrations of sea anemone toxin. These Na⁺ channels revealed by sea anemone toxin are resistant to TTX.
2. Myoblasts prior to fusion are electrically excitable ( \(\dot V\) _max=10 V/s). Electrically activated Na⁺ channels are only blocked by high concentrations of TTX. Titration of TTX resistant Na⁺ channels with a tritiated derivative of TTX indicates a dissociation constant of the TTX-Na⁺ channel complex of 50 nM.
3. Myotubes have both high and low affinity binding sites for TTX (Frelin et al. 1983). Action potentials ( \(\dot V\) _max=100?200 V/s) are only inhibited at high concentrations of TTX. Experiments with rat myoballs indicate that only Na⁺ channels with a low affinity binding site for TTX are functional in voltage-clamp studies. The K_0.5 value for TTX inhibition of the peak Na⁺ current is observed at 70 nM. Spontaneous contractions of myotubes are blocked by TTX with a K_0.5 value of 100 nM, suggesting that TTX resistant Na⁺ channels are also the ones responsible for the spontaneous contractions in rat myotubes in culture.

²²Na⁺ flux studies after activation of the Na⁺ channel with neurotoxins have been carried out at the different stages of differentiation. Toxin activated Na⁺ channels have the same high affinity for sea anemone toxins at all stages of development; likewise, the sensitivity for TTX is the same. The in vitro differentiation of Na⁺ channels in rat and chick skeletal muscle cells is compared and discussed in relation to the action of neurotrophic influences.     

Block of BK (maxi K) channels of rat pituitary melanotrophs by Na+ and other alkali metal ions

The block of large-conductance calcium-activated potassium (BK) channels by internal and external alkali metal ions was studied in adult rat melanotrophs. Internal but not external 20 mM Na⁺ produced a strongly voltage-dependent, flickery block that was well-fitted to the Woodhull model by using a value of 140 mM for the dissociation rate constant at 0 mV [K _d(0)] and an equivalent valence (zδ) of 0.9. At a concentration of 20 mM external K⁺, Cs⁺ and Rb⁺, but not Li⁺, caused a rightward shift of the voltage dependence of the intracellular Na⁺ (Na⁺ _i ) block. This effect of K⁺, Cs⁺ and Rb⁺ was modelled by an equilibrium knock-out mechanism in which the block-relieving ion binds to a site located within the voltage field and consequently increases the off-rate of Na⁺. Internal Li⁺ caused little or no block whereas internal Cs⁺ caused a voltage-dependent block [K _d(0) ≈150 mM]. Flickery channel block observed in cell-attached patches was consistent with a cytoplasmic Na⁺ activity between 1 and 10 mM. Received: 22 January 1996 /Accepted: 26 March 1996     

Effect of amiloride on arachidonic acid and histamine release from rat mast cells

The effect of a putative Na⁺/H⁺ exchange inhibition on histamine and [¹⁴C]arachidonic acid ([¹⁴C]AA) release has been examined in rat peritoneal mast cells, using either addition of amiloride or removal of extracellular Na⁺. The cells were stimulated by non-immunological agents, i.e. calcium ionophore A23187, nerve growth factor (NGF), thapsigargin and compound 48/80. On the basis of the results obtained, a possible role for Na⁺/H⁺ exchange in rat mast cell secretion is discussed.     

Activation of the Na-K pump by intracellular Na in rat slow- and fast-twitch muscle

Experiments were performed on isolated rat soleus (slow-twitch) and extensor digitorum longus (EDL) (fast-twitch) muscle of 4-week-old rats. In soleus muscle, electrical simulation at 2 Hz for 5 min increased the ouabain-suppressible ⁸⁶Rb⁺uptake by 138%, without significant changes in intracellular Na⁺content or Na⁺/K⁺ratio. In EDL muscle, the ouabain-suppressible ⁸⁶Rb⁺uptake was stimulated by only 58%, whereas intracellular Na⁺content and Na⁺/K⁺ratio were increased by around 70%. Na⁺-loading of the muscles by exposure to K⁺-free or K⁺-Ca²-Mg²⁺-free buffer stimulated the ouabain-suppressible ⁸⁶Rb⁺uptake in the two muscles to roughly the same extent, but in EDL muscle this was associated with a more than twofold larger increase in Na⁺/K⁺ratio. When the Na⁺influx was increased by exposure to veratridine similar results were obtained. Graded variation in intracellular Na⁺content was achieved by exposure to monensin. In soleus muscle, a 25% increase in intracellular Na⁺/K⁺ratio resulted in a doubling of the ouabain-suppressible ⁸⁶Rb⁺uptake, whereas a doubling of the Na⁺–K⁺transport rate in EDL muscle required a 140% increase in Na⁺/K⁺ratio. The results indicate that in soleus muscle the Na⁺/K⁺pump is much more sensitive to changes in intracellular Na⁺content than in EDL muscle. This might explain the larger activation of the Na⁺–K⁺pump in slow-twitch muscle during electrical stimulation and might be of significance for the activation of the Na⁺-K⁺pump in vivo during work.