Diverging effects of 5-HT3 receptor antagonists ondansetron and granisetron on estramustine-inhibited cellular potassium transport

We used 86Rb+ (K+ analogue) to study potassium influx during the interaction of highly specific 5-HT3-receptor antagonists, ondansetron and granisetron, with the effects of the anticancer drug, estramustine phosphate, on P31 mesothelioma cells. Estramustine phosphate (80 mg/l, 142 micromol/l) for 120 min. reduced 86Rb+ influx by 18.7%. The reduction was inhibited by ondansetron (0.1 micromol/l), but augmented by granisetron (0.1 micromol/l). Serotonin (1.0 micromol/l) antagonized ondansetron inhibition and restored granisetron-augmented reduction of estramustine phosphate-induced 86Rb+ influx to the level of the drug itself. Estramustine phosphate inhibited cellular Na+, K+, 2Cl- -cotransport activity whereas Na+, K+, ATPase activity was unaffected. Ondansetron blockade of estramustine phosphate-induced reduction of 86Rb+ influx was due to increased Na+, K+, ATPase and Na+, K+, 2Cl- -cotransport whereas augmentation of estramustine phosphate-induced reduction of 86Rb+ influx by granisetron, or combination of 5-HT3 receptor antagonists with serotonin was due mainly to inhibition of cellular Na+, K+, ATPase activity Thus, ondansetron possesses a distinct ability to reverse K+ influx of tumour cells exposed to estramustine phosphate whereas granisetron does not, due to different effect on cellular Na+, K+, ATPase and Na+, K+, 2Cl- -cotransport activity. Highly 5-HT3 receptor-specific antiemetic agents may have different effects on ion transport of tumour cells during treatment with cytotoxic drugs.     

Chronology of patulin-induced alterations in membrane function of cultured renal cells, LLC-PK

In a previous study we compared the effects of patulin (PAT) and ouabain, a specific inhibitor of the Na(+)-K+ ATPase, and found significant differences with regard to the kinetics of Na+ influx and K+ efflux, and sulfhydryl reactivity in LLC-PK1 cells. The purpose of the present study was to determine the relationship between Na+ influx, K+ efflux, membrane potential ([3H]tetraphenylphosphonium accumulation), cellular viability [lactate dehydrogenase (LDH) release], and changes in cell morphology (blebs). The effects of PAT are concentration and time dependent. At concentrations of PAT above 10 microM there is a transient increase in intracellular electronegativity (less than 1 hr) followed by a sustained depolarization (greater than 1 hr) which is correlated with complete Na+ influx, K+ efflux, total LDH release, and bleb formation. However, at PAT concentrations of 5-10 microM there is a sustained increased intracellular electronegativity (4-8 hr) which is associated with partial Na+ influx and K+ efflux, no significant LDH release, and relatively few blebs. The hyperpolarizing effect may be a result of increased permeability to K+ relative to Na+. At times and concentrations which result in increased intracellular electronegativity, PAT has no effect on [3H]ouabain binding and thus increased Na+/K+ pump turnover does not seem to be the cause of the transient hyperpolarizing effect of PAT. These results are consistent with the hypothesis that PAT causes alterations in plasma membrane permeability which favor K+ efflux relative to Na+ influx. The toxic effects of PAT are irreversible in LLC-PK1 cells after even short pretreatment with PAT. The primary toxic lesion appears to be at some level other than that involving inhibition of macromolecular synthesis, perhaps the plasma membrane itself.     

N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) stimulation of K+ transport in a human salivary epithelial cell line.

Treatment of a human salivary epithelial cell line, HSG-PA, with the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7; 20-70 microM) increased 86Rb (K+) influx and efflux in a manner similar to that resulting from muscarinic (carbachol; Cch) or calcium ionophore (A23187) stimulation. Unlike the Cch or A23187 responses, the W7 responses were not blocked by 0.1 mM atropine (muscarinic antagonist) or phorbol-12-myristate-13-acetate (0.1 microM). Like Cch- or A23187-stimulated 86Rb fluxes, W7-stimulated 86Rb fluxes were substantially blocked by the K+ channel inhibitors quinine (0.25 mM) and scorpion venom-containing charybdotoxin (33 micrograms/mL), while 5 mM tetraethylammonium chloride (K+ channel blocker), furosemide (0.1 mM; Na+,K+,2Cl- co-transport inhibitor) and ouabain (10 microM; Na+,K(+)-ATPase inhibitor) were ineffective. Purified charybdotoxin (10 nM) also blocked W7-stimulated 86Rb influx, as well as 86Rb influx stimulated by Cch or A23187. Although Quin 2 fluorescence measurements indicated that W7 increased free intracellular Ca2+ concentration ([Ca2+]i), the magnitude of the increase appeared to be insufficient to solely account for the W7-stimulated increases in 86Rb fluxes (i.e. K+ channel activity). Ca2+ was involved in the W7 response, however, as lack of Ca2+ in the incubation medium reduced the W7-stimulated increases in 86Rb influx and efflux. Taken together, our results suggest that W7 increased K+ fluxes in HSG-PA cells by interacting, directly or indirectly, with the K+ transport machinery (K+ channels) in a manner different from that observed during muscarinic stimulation, and also in a manner not accounted for solely by the formation of a typical muscarinic- or calcium ionophore-generated calcium signal.     

THE INFLUENCE OF A HIGH K INTAKE ON THE HYPERTENSIVE EFFECT OF ACTH AND DOC IN SHEEP

The effect of 5 days administration of ACTH or DOC, was examined before and after chronic potassium (K) loading in sheep. K loading raised plasma [K], urine volume and K excretion but had no effect on mean arterial pressure (MAP). On a normal K intake DOC (5 mg/day i.v.) increased MAP and plasma sodium [Na]. Plasma [K], urinary Na (day 1) and K (day 1) excretion were decreased. On a high K intake (congruent to 800 mmol K/day), DOC lowered plasma [K] but had no effect on MAP or Na excretion. The hypertensive effects of ACTH were not affected by K intake.     

The effects of muzolimine and urine from muzolimine-treated rats on Na+K+Cl- cotransport in Madin-Darby canine kidney cells.

Muzolimine is a loop diuretic with an original chemical structure devoid of the acidic or sulfonamide group known to be necessary for an interaction with Na+K+Cl- cotransport. We studied the effects of urine from muzolimine-treated rats on the Na+K+Cl- cotransport-dependent 86Rb influx in MDCK cells. Na+K+Cl- cotransport was inhibited by urine obtained 15 min (42% inhibition) and 60 min (49% inhibition) after muzolimine injection (50 mumol/kg i.v.). Muzolimine itself was not detectable in the urine. Probenecid (100 mumol/kg i.v.) suppressed both the diuretic effect of muzolimine and the inhibition of Na+K+Cl- cotransport by urine from muzolimine-treated rats. These results suggest that the diuretic effect of muzolimine is due to the metabolism of muzolimine into an active compound which inhibits Na+K+Cl- cotransport after its secretion into the tubular lumen via a proximal pathway. The direct effect of muzolimine on Na+K+Cl- cotransport in MDCK cells was also tested: surprisingly, the inhibition of 86Rb influx was significant in the presence of muzolimine (IC50 = 1.44 microM). We show that this effect was due to the metabolism of muzolimine by these cells into an active compound.     

Estimation of the ionic mechanism of inotropic agents from the shifting pattern of frequency-force relationship in guinea pig ventricular myocardium

Experiments were carried out to get information about the shifting mode of the frequency-force relationship curve (FFRC) of several well-established agents in guinea pig ventricular myocardium to estimate the ionic mechanism of inotropic action. The increase in [Ca2+]o or decrease in [Na+]o shifted the FFRC to the left and upwards, but no shift was observed when [Ca2+]o/[Na+]o2 ratio was kept constant. Drugs increasing [Na+]i (veratridine, asebotoxin) caused a parallel leftward shift of FFRC in the middle range of contraction frequency. Tetrodotoxin depressed the force of contraction (Fc) at higher frequencies, reflecting the fast Na influx inhibition. Nifedipine depressed, while BAY K 8644 augmented, the Fc at all frequency ranges, reflecting the Ca influx modulation. Ouabain and [c-AMP]i-elevating agents (isobutyl methylxanthine, histamine) strengthened the Fc at all frequencies. The ionic mechanism of these two groups of cardiotonic agents depending primarily on either Na or Ca influx could be estimated from a distinct time difference of the peak in the "rested-state" contraction curves of guinea pig papillary muscle. The shifting pattern of FFRC will contribute to the estimation of the mechanism of inotropic agents.     

Elevated extracellular K(+) concentrations inhibit N-methyl-D-aspartate-induced Ca(2+) influx and excitotoxicity.

Although extracellular [K(+)] ([K(+)](E)) is highly elevated during brain ischemia, in vitro studies aimed at explaining the mechanisms of excitotoxicity have been conducted at low [K(+)](E). Whether high [K(+)](E) affects excitotoxicity has not been formally addressed. Therefore this study, using digital fluorescence microscopy, tested how the elevation of [K(+)](E) from 5.6 to 60 mM affects N-methyl-D-aspartate (NMDA)-induced Ca(2+) and Na(+) influx, plasma membrane (PM) potential, mitochondrial Ca(2+) load, and viability of primary cultures of rat cerebellar granule cells. High [K(+)](E) curtailed the NMDA-induced Ca(2+) and Na(+) influx and mitochondrial Ca(2+) overload, and prevented neuronal death. Surprisingly, the inhibitory effect of high [K(+)](E) on the NMDA-induced Ca(2+) influx could not be linked to depolarization of the PM. Apparently, the PM of cerebellar granule cells exposed to NMDA was more depolarized at low than at high [K(+)](E), probably because the NMDA-induced Na(+) influx was greatly enhanced when the extracellular [Na(+)]/[K(+)] ratio was increased. When this ratio was small, i.e., at high [K(+)](E), the NMDA-induced increase in cytoplasmic [Na(+)] was suppressed, preventing Ca(2+) influx via the reverse operation of the Na(+)/Ca(2+) exchanger, which may explain the inhibitory effect of high [K(+)](E) on NMDA-induced Ca(2+) influx and excitotoxicity.     

14C]guanidinium ion influx into Na+ channel preparations from mouse cerebral cortex

[14C]Guanidinium ion influx into Na+ channel preparations from mouse and rat cerebral cortex (purified synaptosomes, and synaptoneurosomes) was characterized and its properties were compared with those for 22Na+ influx. Tetrodotoxin-sensitive influx of [14C]guanidinium ion was stimulated by aconitine, veratridine, and batrachotoxin with a K0.5 of 7, 5 and 0.3 microM, respectively, the maximal influx being the same with all toxins. Scorpion venom shifted the activation curve of veratridine to the left, but did not increase the maximal influx. The potency of the local anesthetic drugs cocaine and tetracaine in inhibiting [14C]guanidinium ion influx depended upon the concentration of veratridine used to activate the Na+ channels. The mechanism of inhibition was of a competitive nature. Other local anesthetic drugs and cocaine congeners inhibited [14C]guanidinium ion influx with potencies very similar to those for inhibition of 22Na+ influx. The results show that [14C]guanidinium ion influx is a valid model for 22Na+ influx through voltage-dependent Na+ channels although there are some differences between the two influx assays. The guanidinium ion assay offers the convenience of the 14C isotope as compared with the strongly radiating 22Na+ isotope.     

前胡香豆素对肾型高血压大鼠左室肥厚及心肌胞内钙、Na+,K+-ATP酶和Ca2+,Mg2+-ATP酶活性的影响

目的研究前胡香豆素组分对肾型高血压左室肥厚的预防和逆转作用及机制。方法用两肾一夹肾型高血压左室肥厚大鼠(RHR)模型,测定前胡香豆素组分对其血压、左室湿重、心肌细胞面积、胞内静息钙及胞膜和线粒体ATP酶活性的影响。结果前胡香豆素组分(30 mg·kg^-1·d^-1,ig)预防组及逆转组大鼠血压、左室湿重/体重均较肥厚组明显降低;左室心肌细胞面积、胞内静息钙均较肥厚组降低;对KCl致钙浓度升高亦明显低于肥厚组;两组均可增加心肌细胞膜及线粒体Na⁺,K⁺-ATP酶和Ca²⁺,Mg²⁺-ATP酶活性。结论前胡香豆素组分可预防及逆转RHR左室肥厚,减少心肌细胞内钙含量,增加ATP酶活性。     

The acute in vitro effect of ethanol, its metabolites and other toxic alcohols on ion flux in isolated human leucocytes and erythrocytes

Using isolated healthy human leucocytes and erythrocytes as model cells, we investigated the inhibitory effect of ethanol, its metabolites and of other toxic alcohols on the active fluxes of rubidium (Rb: equivalent to K) and sodium (Na), and on Na,K-ATPase activity. Ethanol (80 mmol X l-1) inhibited total and ouabain-sensitive 86Rb influx and 22Na efflux in leucocytes, this being dose-related for total, ouabain-sensitive and ouabain-insensitive fluxes at higher concentrations. In erythrocytes inhibition occurred at 20 mmol X l-1 for 86Rb influx, dose-related at higher concentrations as for leucocytes. 22Na efflux was inhibited at 80 mmol X l-1 and above. Acetaldehyde (0.1 and 0.2 mmol X l-1), 1,2-propanediol (0.8 mmol X l-1) and 2,3-butanediol (0.4 mmol X l-1) inhibited all fractions of 86Rb influx in erythrocytes, but not in leucocytes. Methanol, 2-propanol and 1,2-ethanediol (16 and 32 mmol X l-1) inhibited 86Rb influx in erythrocytes, but not in leucocytes. The order of potency was 2-propanol greater than 1,2-ethanediol greater than methanol. Na,K-ATPase activity was inhibited in lysed leucocyte and erythrocyte preparations only at very high concentrations of the alcohols--suggesting that inhibition is due to an alteration in membrane structure and not to a direct effect on the enzyme.     

Ethanol effects on active Na+ and K+ transport in cultured fetal rat hepatocytes

To define further the influence of ethanol on membranes, its effects on Na+ pump function were studied in monolayer cultures of fetal rat hepatocytes. The effects of ethanol (2 and 4 mg/ml) on total K+ influx, ouabain-sensitive K+ influx, Na+ pump density (from specific [3H]ouabain binding), pump turnover rates and intracellular Na+ were measured following exposure of the cells to ethanol for 1-24 hr. In parallel studies, the effects of ethanol (2 mg/ml) on cell water content and membrane fluidity were measured. Ethanol had no immediate effect on K+ influx, but after 1 hr ethanol in concentrations of 2 and 4 mg/ml decreased the total K+ influx (mumol/10(11) cells/sec) from a control of 8.5 +/- 0.64 to 4.46 +/- 0.50 and 4.09 +/- 0.26 respectively (N = 6 for each experiment; P less than 0.001). This represented the maximum effect of ethanol since after 6 and 24 hr of ethanol treatment the K+ influx had increased towards control levels but remained significantly (P less than 0.01 for 2 mg/ml and P less than 0.001 for 4 mg/ml) below that in control cells even at 24 hr. The decrease in K+ influx reflected a decrease in mean ouabain-sensitive K+ influx from a control of 5.87 to 3.24 and 2.70 (mumol/10(11) cells/sec) after a 1-hr treatment with 2 and 4 mg ethanol/ml medium respectively. Ethanol (2 mg/ml) treatment for 1-hr decreased Na+ pump density (x 10(5) molecules ouabain per cell) from a control of 2.80 +/- 0.30 to 1.70 +/- 0.11 (P less than 0.001). At 6 and 24 hr [3H]ouabain binding showed a pattern similar to that seen with the K+ influx, tending to return to pretreatment levels. There was no change in individual pump turnover rates in the presence of ethanol. Following exposure to ethanol, cellular Na+ content steadily increased over the first 6 hr and then returned to control levels. When corrected for parallel changes in cell volume, however, intracellular Na+ concentration increased by 17% (P less than 0.01) after 1 hr and thereafter remained at this higher level throughout the 24-hr period. Measurements of membrane fluidity showed that it was increased markedly by ethanol at a concentration of 2 mg/ml and that the effect bore a close temporal relationship to the changes in active K+ influx and Na+ pump density. We conclude that ethanol has a depressant effect on hepatic Na+ pump function, resulting in an increase in intracellular Na+ and an eventual gain in cell water.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of amiloride and its analogues on [3H]batrachotoxinin-A 20-alpha benzoate binding, [3H]tetracaine binding and 22Na influx

The ability of amiloride and its analogues to inhibit [3H]batrachotoxinin-A 20-alpha benzoate [( 3H]BTX-B) and [3H]tetracaine binding to rat synaptosomes and to a rat heart membrane preparation was tested. Their ability to inhibit 22Na influx was determined with rat synaptosomes. 5-N-substituted analogues were generally more potent in inhibiting [3H]BTX-B and [3H]tetracaine binding than compounds substituted on the guanidine group. However, the inhibition was not competitive. Amiloride and some of its analogues were as active or more active in inhibiting [3H]tetracaine binding than they were in inhibiting [3H]BTX-B binding. 22Na influx was inhibited with the same relative potencies as [3H]BTX-B binding and a good correlation was found between the two inhibitions. These results show an effect of amiloride and its analogues on the voltage-sensitive Na+ channels, which could partly explain the inotropic effects of these drugs.     

Effects of glutamate transport substrates and glutamate receptor ligands on the activity of Na-/K(+)-ATPase in brain tissue in vitro

1. It has been suggested that Na+/K(+)-ATPase and Na(+)-dependent glutamate transport (GluT) are tightly linked in brain tissue. In the present study, we have investigated Na+/K(+)-ATPase activity using Rb+ uptake by 'minislices' (prisms) of the cerebral cortex. This preparation preserves the morphology of neurons, synapses and astrocytes and is known to possess potent GluT that has been well characterized. Uptake of Rb+ was determined by estimating Rb+ in aqueous extracts of the minislices, using atomic absorption spectroscopy. 2. We determined the potencies of several known substrates/inhibitors of GluT, such as L-trans-pyrrolidine-2,4-dicarboxylate (LtPDC), DL-threo-3-benzyloxyaspartic acid, (2S,3S,4R)-2-(carboxycyclopropyl)-glycine (L-CCG III) and L-anti,endo-3,4-methanopyrrolidine dicarboxylic acid, as inhibitors of [3H]-L-glutamate uptake by cortical prisms. In addition, we established the susceptibility of GluT, measured as [3H]-L-glutamate uptake in brain cortical prisms, to the inhibition of Na+/K(+)-ATPase by ouabain. Then, we tested the hypothesis that the Na+/K(+)-ATPase (measured as Rb+ uptake) can respond to changes in the activity of GluT produced by using GluT substrates as GluT-specific pharmacological tools. 3. The Na+/K(+)-ATPase inhibitor ouabain completely blocked Rb+ uptake (IC50 = 17 micromol/L), but it also potently inhibited a fraction of GluT (approximately 50% of [3H]-L-glutamate uptake was eliminated; IC50 < 1 micromol/L). 4. None of the most commonly used GluT substrates and inhibitors, such as L-aspartate, D-aspartate, L-CCG III and LtPDC (all at 500 micromol/L), produced any significant changes in Rb+ uptake. 5. The N-methyl-D-aspartate (NMDA) receptor agonists (R,S)-(tetrazol-5-yl)-glycine and NMDA decreased Rb+ uptake in a manner compatible with their known neurotoxic actions. 6. None of the agonists or antagonists for any of the other major classes of glutamate receptors caused significant changes in Rb+ uptake. 7. We conclude that, even if a subpopulation of glutamate transporters in the rat cerebral cortex may be intimately linked to a fraction of Na+/K(+)-ATPase, it is not possible, under the present experimental conditions, to detect regulation of Na+/K(+)-ATPase by GluT.     

Cellular potassium ion deprivation enhances apoptosis induced by cisplatin

The anticancer drug cisplatin induces cell death by apoptosis. Apoptosis is dependent on cellular loss of potassium ions (K+). We have recently shown that the antifungal drug amphotericin B (enhancing K+ efflux), combined with the Na+, K+, 2Cl(-)-cotransport blocker bumetanide (decreasing K+ influx), augmented cisplatin-induced apoptosis in vitro. We therefore quantified K+ fluxes with the K+ analogue rubidium (86Rb+) in cisplatin-induced apoptosis of mesothelioma cells treated with bumetanide and amphotericin B. Bumetanide combined with amphotericin B enhanced cisplatin-induced apoptosis by a pronounced initial reduction of K+ influx due (in addition to Na+, K+, 2Cl(-)-cotransport inhibition) also to Na+, K+, ATPase pump inhibition. As 86Rb+ efflux was initially preserved, combination of the drugs would lead to net K+ loss. Combinations of K+ flux modulators leading to cellular potassium ion deprivation thus augments cisplatin-induced apoptosis and could therefore possibly be used to enhance the antitumour efficacy of cisplatin treatment.     

INFLUENCE OF AGE, SEX AND WEIGHT ON MEASUREMENTS OF INTRACELLULAR Na+ AND K+ IN ESSENTIAL HYPERTENSION

Na+ and K+ concentrations were measured in red cells from 22 patients with untreated essential hypertension and 22 normotensive control subjects who were individually and prospectively matched for age, sex and weight and in a separate population of 31 hypertensive patients and 41 normotensive subjects who were not so matched. Intracellular Na+ and K+ concentrations were similar in the matched subjects. Significant positive correlations were found between intracellular Na+ and body weight (r = 0.27, P less than 0.041), and between intracellular K+ and age (r = 0.29, P less than 0.028). In the unmatched population, intracellular Na+ was significantly increased in patients with hypertension and was correlated significantly with age (r = 0.25, P less than 0.017). After correction for age, intracellular Na+ was similar in both hypertensive and normotensive subjects. Age-corrected intracellular K+ was similar in both hypertensive and normotensive groups but was significantly lower in male subjects. These results highlight the importance of matching study groups for age, sex and weight when studying possible abnormalities in cell ionic composition in hypertension. False positive results may arise if study groups are not matched for these characteristics. Red cell Na+ and K+ are not altered in essential hypertension.     

Role of Na+/H+ exchanger in acetylcholine-mediated pulmonary artery contraction of spontaneously hypertensive rats

Compared to sympathetic nervous system, the role of parasympathetic innervation on tone development, especially under diseased conditions, of the pulmonary artery is relatively unknown. In this study, the contractile effect of acetylcholine and the type(s) of muscarinic (M) receptor involved in the pulmonary artery (1st intralobar branch; endothelium-denuded, under resting tension) of the normotensive Wistar-Kyoto (WKY) and age-matched (male, 22-26 weeks old) Spontaneously hypertensive rats (SHR) were investigated. Cumulative administration of acetylcholine (> or =0.1 microM) caused a concentration-dependent increase in tension (antagonised by p-fluoro-hexahydro-sila-difenidol and 4-diphenylacetoxy-N-methylpiperidine, both are selective muscarinic M(3) receptor antagonists) and the magnitude of maximum contraction (expressed as % of 50 mM [K(+)](o)-induced contraction) was markedly enhanced in the presence of neostigmine (10 microM, an anti-cholinesterase) (acetylcholine 30 microM, SHR: 72% vs. 35%; WKY: 32% vs. 20%). In SHR only, acetylcholine-elicited contraction was suppressed by 1-[beta-[3-(4-Methoxyphenyl)-propoxyl]-4-methoxyphenethyl]-1H-imidazole (SK&F 96365, 1 microM), amiloride (500 microM), ethyl-isopropyl-amiloride (EIPA, 10 microM), 2-[2-[4-(4-Nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R 7943, 5 microM), 2,4-dichlorobenzamil (10 microM), and an equal molar substitution of [Na(+)](o) (< or =30 mM) with choline or N-methyl-D-glucamine. In nominally [Ca(2+)](o)-free, EGTA (0.5 mM)-containing Krebs' solution, acetylcholine (> or =3 microM) only elicited a small contraction. In conclusion, muscarinic M(3) receptor activation is responsible for the pulmonary artery contraction induced by acetylcholine, with a greater magnitude observed in SHR. The exaggerated contraction in SHR is probably due to an influx of [Na(+)](o) through the Na(+)/H(+) exchanger and the store-operated channels (SOC) into smooth muscle cells. Elevation of cytosolic [Na(+)](i) subsequently leads to an influx of [Ca(2+)](o) through the reverse mode of the Na(+)/Ca(2+) exchanger seems to play a permissive role in mediating the exaggerated contractile response of acetylcholine recorded in the SHR.     

Inhibition by cations of antagonist binding to histamine H1-receptors: differential effect of sodium ions on the binding of two radioligands.

1. Measurements have been made of the inhibition by mono- and divalent cations of the binding of [3H]-(+)-N-methyl-4-methyldiphenhydramine ([3H]-QMDP) to histamine H1-receptors in homogenates of guinea-pig cerebellum. 2. The binding of [3H]-QMDP was inhibited by monovalent cations with an order of potency Li+ = Na+ greater than K+ greater than Cs+ = Rb+. The IC50 for Li+ was 39 mM, but that for K+ was 132 mM. Hill coefficients for inhibition curves for Li+ and Na+ were less than 1. 3. Divalent cations also inhibited the binding of [3H]-QMDP. The most potent cations examined were Hg2+, Cd2+ and Zn2+, with IC50 values of 5, 17 and 41 microM, respectively. Ca2+ and Mg2+ were relatively weak inhibitors (IC50 12 and 34 mM, respectively). The potency of Ni2+, Co2+ and Mn2+ was intermediate between these groups. Hill coefficients for inhibition curves for Hg2+, Cd2+ and Zn2+ were greater than 1, but Hill coefficients for the other cations were less than 1. 4. Both mono- and divalent cations also inhibited the binding of [3H]-mepyramine. The divalent cations were approximately equipotent in inhibiting the binding of [3H]-QMDP and [3H]-mepyramine. The same was true for Li+. However, Na+ was markedly more effective against [3H]-QMDP binding than against the binding of [3H]-mepyramine. 5. The effect of 40 mM Li+ on the parameters of binding of [3H]-mepyramine was to increase the best-fit value of the concentration giving half-maximal binding EC50, by approximately 2 fold without having any significant effect on the maximum amount of binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of sodium ion influx in depolarization-induced neuronal cell death by high KCI or veratridine.

Intracellular Na+ concentration plays an important role in the regulation of cellular energy metabolism; i.e., increased intracellular Na+ concentration stimulates glucose utilization both in cultured neurons and astrocytes. Both high KCI and veratridine, which have been known to cause neuronal damage, elicit increased glucose utilization, presumably via increased intracellular Na+ concentration. In the present study, we examined the role of intracellular Na+ influx in the mechanisms of neuronal cell damage induced by high KCl or veratridine assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric method. Rat primary cultures of striatal neurons were incubated with high KCl (final concentrations: 25, 50 mM) or veratridine (0.1-100 microM) with or without various inhibitors. High KCl depolarizes cell membrane, thus, leading to Na+ influx through an activation of voltage-sensitive Na+ channels, while veratridine elicits Na+ influx by directly opening these channels. After 24-h incubation with elevated [K+]o or veratridine, glucose contents in the medium decreased significantly (approximately by 7 mM), but remained higher than 18 mM. High [K+]o reduced percent cell viability significantly (approximately 50% at 25 mM, approximately 40% at 50 mM [K+]o, P<0.01), but tetrodotoxin (100 nM) had no protective effect, indicating that Na+ influx was not essential to high K+ -induced cell death. DL-2-Amino-5-phosponovaleric acid (APV) (1 mM) completely blocked cell death induced by elevated [K+]o, while 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 microM) did not. In contrast, veratridine (>10 microM) caused cell damage in a dose-dependent and tetrodotoxin-sensitive manner, but none of APV, CNQX, or bepridil (Na+ -Ca2+ exchanger blocker) had any protective effect. Nifedipine (50 approximately 100 microM), however, reduced percent cell damage induced by veratridine.     

Decreased Na+-K+-ATPase activity and [3H]ouabain binding sites in various tissues of spontaneously hypertensive rats

Na+-K+-ATPase activity and [3H]ouabain binding were studied in cerebral cortex, kidney and heart isolated from spontaneously hypertensive rats (SHR) in both the prehypertensive (6 week old) and the hypertensive stages (14 week old). Na+-K+-ATPase activity of heart and kidney was found to be decreased by about 38 and 16% in the prehypertensive and hypertensive stages of SHR respectively; that of cerebral cortex decreased by 23.5% only in the hypertensive stages. Similar results were obtained by pretreatment of membranes with either 0.001% Triton X-100 or by increasing the K concentration from 4.7 to 12.7 mM in the Krebs solution. No significant differences in microsomal protein yield were noted between prehypertensive or hypertensive SHR and the age-matched WKY rats. The study of binding of [3H]ouabain to cerebral cortex, kidney and heart showed that the decreased Na+-K+-ATPase in hypertensive SHR was due to a 31.6, 21.8 and 41.3% reduction in the number of high affinity binding sites respectively, while the affinity constants (Kd) of ouabain binding sites on this enzyme in cerebral cortex, kidney and heart of the normotensive WKY rats were 26.5, 455.9 and 74.7 nM respectively and those from the hypertensive SHR were not altered. The plasma K concentration of the SHR in the prehypertensive and hypertensive stages was 4.07 and 4.13 mM, respectively, significantly less than that of the age-matched WKY rats. It appears that the decrease of plasma K and Na+-K+-ATPase activity in heart and kidney in SHR is derived from a genetic defect and may be related to the abnormal Na handling in this genetically hypertensive strain.     

Binding properties and biological effects of oxidized-ouabain on cultured neonatal-rat cardiac myocytes. Implications on the mechanism of action of the digitalis-glycosides.

Mild oxidation of ouabain with NaIO4, causes the cleavage of the bond between C2' and C3' of the rhamnose ring, leaving the steroid moiety intact. The oxidized ouabain (ox-ouabain) was examined on spontaneously contracting cultured rat-cardiac myocytes. Two classes of binding sites, with high and low affinities, were detected for both ox-ouabain and unmodified ouabain. The dissociation constants (KD) were found to be similar for both compounds, but the rate constants of association (ka) and dissociation (kd) of the low affinity sites were higher for ox-ouabain as compared with ouabain. Displacement experiments showed that ox-ouabain and ouabain bind to the same sites. The effects of ox-ouabain and ouabain on the activity of Na+, K(+)-ATPase were determined in microsomal preparations. Similar dose-response curves for the inhibition of the enzyme activity were determined for both drugs. Inhibition was observed only at concentrations above 10(-6) M. The biological effects of the drugs were examined by their capacity to induce positive inotropic or toxic effects. Concentrations of ox-ouabain which induced positive inotropic effects (increase in amplitude of systolic cell motion), ranged from 5 x 10(-8) M to 5 x 10(-6) M, as compared with 10(-7) M to 5 x 10(-7) M with ouabain. "Toxic" effects (decrease in the amplitude of systolic motion, increased beating frequencies and elevation in the position of maximal relaxation) was observed only with 10(-5) M ox-ouabain as compared with 10(-6) M ouabain. The mechanism of the inotropic action of ox-ouabain at the lower concentration range was investigated by measuring the effect of the drugs on 86Rb+ (analogue of K+) influx. Dose-response curves of effects of ouabain and ox-ouabain on 86Rb+ influx were bi-phasic. At low concentrations stimulation was observed, whereas at high concentrations 86Rb+ influx was inhibited. Ox-ouabain stimulated 86Rb+ influx by lower concentrations and to a greater extent than ouabain. A part of 86Rb+ influx into cardiac myocytes is mediated by the K+/Na+/Cl- cotransporter, which can be inhibited by loop diuretic drugs such as bumetanide. We have previously shown that ouabain, at low concentrations, stimulates the activity of the cotransporter. It is shown in the present work that ox-ouabain stimulates the activity of the cotransporter by lower concentrations and to a greater extent than ouabain.(ABSTRACT TRUNCATED AT 400 WORDS)