Platelet Ca2+ homeostasis: Na+-Ca2+ exchange in plasma membrane vesicles.

A vesicular plasma membrane-enriched fraction obtained from human platelets exhibited 45Ca2+ uptake in exchange for intravesicular Na+. The rate of Ca2+ uptake was linear up to 4 sec. The apparent Km for Ca2+ was 22 microM and the Vmax 280 pmol/mg/sec. Ca2+ efflux from Ca2+ loaded vesicles was obtained upon dilution into a NaCl but not a KCl medium. The extent of Ca2+ uptake was monotonically increased as the pH increased from 6 to 9. Na+-Ca2+ exchange was shown to be electrogenic. Ca2+ uptake was distinguished from binding by the induction of Ca2+ release after A23187 addition. These findings support a role for Na+-Ca2+ exchange in human platelet Ca2+ transport.

The action of thiamine and its di- and triphosphates on the slow exponential decline of the ionic currents in the node of Ranvier.

Sodium and potassium currents in the node of Ranvier decrease exponentially with time during long lasting voltage clamp experiments. This decline is strongly dependent on temperature (Q10 approximately 3). Thiamine and, particularly, its diand triphosphoric acid esters are shown to prevent this exponential decline of the ionic currents. Thiamine acts from the outside and from the inside of the nodal membrane, but more potently from the inside. Thiamine diphosphate prevents the exponential decline of the ionic currents only when applied internally. Thiamine triphosphate, the most effective thiamine derivative was tested form the inside only. Bacterial thiaminases applied externally were not effective, presumably because they do not permeate the nodal membrane. Tetrodotoxin, that has been shown by other investigators to induce a release of thiamine from nerve membranes, does not alter the action of thiamine on the exponential decline of current and vice versa. It is concluded that: (1) thiamine diphosphate or thiamine triphosphate are the active thiamine compounds in nerve membranes; (2) the site of action is located at the internal suface of the membrane; (3) the reduction of the thiamine concentration in the membrane or in the axoplasm could cause the exponetial decline of currents; (4) the release of thiamine from nerve membranes induced by tetrodotoxin is interpreted as a side effect not even related to the mechanism by which tetrodotoxin blocks the sodium channels; (5) thiamine polyphosphates appear to stabilise the intrinsic electric field strength of the nodal membrane in the resing state. Threfore, as a working hypothesis, it is suggested that the thiamine derivatives control the number of functioning ionic channels by stabilising the density of negative surface charges at the inner side of the nerve membrane.

pH gradient-stimulated sulfate transport by rabbit ileal brush-border membrane vesicles: evidence for SO4-OH exchange.

In the presence of a pH gradient (7.7 inside, 5.5 outside), the initial velocity of SO4 uptake by rabbit ileal brush-border membrane (BBM) vesicles was markedly stimulated compared with uptake in the absence of a pH gradient. Under pH gradient conditions, SO4 was transiently accumulated at a concentration 13-fold higher than at equilibrium ("overshoot"). Superimposition of a HCO3 gradient did not further stimulate the initial velocity of SO4 uptake compared with a pH gradient alone. Evidence that this pH gradient-stimulated SO4 uptake represented SO4-OH exchange included lack of sensitivity of SO4 transport to alterations of the membrane potential; 85-95% inhibition of SO4 uptake by the anion exchange inhibitors DIDS and SITS; and saturation kinetics (Km for SO4 = 0.475 +/- 0.054 mM; Vmax = 4.1 +/- 0.1 nmol SO4 X mg prot-1 X min-1). Sulfate did not inhibit pH gradient-stimulated 36Cl uptake, indicating that SO4-OH and Cl-HCO3(OH) are different exchangers. When BBM vesicles were compared with basolateral membrane (BLM) vesicles, pH gradient-stimulated SO4 uptake was found predominantly in the BBM preparation. Brush-border SO4-OH exchange was further localized by demonstrating Na-stimulated SO4 efflux from vesicles loaded under pH gradient conditions, suggesting that Na-SO4 cotransport and SO4-OH exchange are on the same BBM vesicles. In conclusion, a SO4-OH exchanger (or H-SO4 cotransporter) exists on the brush border of rabbit ileum which is distinct from the brush-border Cl-HCO3(OH) exchanger.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in inositol polyphosphate-sensitive calcium exchange in aortic smooth muscle cells in vitro.

Cells that expressed the muscle-specific intermediate filament protein desmin were cultured from the aorta of Fischer 344 rats. When the cultured cells were extracted with digitonin, they accumulated 45Ca2+ from the incubation medium in a manner that was stimulated by ATP and released subsequently by exposure to the Ca2+ ionophore A23187. Ca2+ bound in the presence of ATP was also released by exposure to inositol 1,4,5-trisphosphate (IP3). Like contraction in some kinds of smooth muscle, IP3 released Ca2+ in either the absence or the presence of the ATPase-inhibitor ruthenium red. When the responsiveness of digitonin-extracted cells cultured from 3-, 12-, and 24-month-old rats was compared, cells from the youngest group released only about one-half as much Ca2+ as cells from the 12- or 24-month-old rats. The results suggest that in the rat there are changes during maturation in the responsiveness to inositol polyphosphates of intracellular compartments that sequester Ca2+ for stimulus-contraction coupling in the aortic smooth muscle cell. These changes, characterized in smooth muscle cells in vitro, might contribute to the way vascular responsiveness is regulated in vivo.

Decamethonium and serum potassium in man.

Decamethonium and succinylcholine were used to study the effects of depolarizing muscle relaxants on serum potassium in 60 patinets, free of neuromuscular disease, during major orthopedic surgery. Significant increases in serum K+ were found after administration of decamethonium or succinylcholine in the usual clinical doses. The abnormal elevations of serum K+ found in patients with burns, massive trauma, or muscle denervation are thus accentuations of the process that occurs in normal man following use of these depolarizing drugs. The administration of any depolarizing agent to these abnormal patient groups would, therefore, appear contraindicated.

NBD-taurine fluorescence as a probe of anion exchange in gallbladder epithelium.

Previous work has demonstrated that after osmotic shrinkage of Necturus gallbladder epithelial cells, their volumes are restored to control levels despite the continued presence of the hyperosmotic medium. It has been proposed that activation of parallel neutral Na+-H+ and Cl--HCO-3 exchangers in the apical membrane is necessary for regulatory volume increase. As an independent technique to determine whether and for how long ion flux through the anion exchanger is actually enhanced by exposure to hypertonicity, fluorescence measurements of N-(2-aminoethylsulfonate)-7-nitrobenz-2-oxa-1,3-diazole (NBD-taurine), a substrate of the anion exchanger in red blood cells, have been made in intact Necturus gallbladder. The cells were loaded with the dye by incubation. The tissue was perfused in a miniature chamber placed on the stage of a microscope and viewed with high-magnification optics combined with video. Fluorescence was monitored at frequent intervals with a photomultiplier tube, and transmittance of the tissue to the laser excitation light was monitored with a photodiode. The epithelium was simultaneously observed with transmitted light to control for changes in focus or lateral movement. Exposure of the tissue to a mucosal medium made hypertonic by the addition of mannitol transiently enhanced the efflux of NBD-taurine from the cells in approximately 70% of the tissues examined. In the presence of the anion-exchange inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS, 100 microM), hypertonicity enhanced NBD-taurine efflux in only 14% of the preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of cations on pH gradient-stimulated sulfate transport in rabbit ileal brush-border membrane vesicles.

In brush-border membrane vesicles from rabbit ileum, we previously reported pH gradient-stimulated SO4 uptake and presented evidence that this represents carrier-mediated SO4-OH exchange. In the present study inhibitors of SO4-OH exchange (H-SO4 cotransport) were shown not to inhibit Na-SO4 cotransport, suggesting that these are two separate carrier-mediated transport mechanisms. While pH gradient-stimulated SO4 uptake was inhibited 87% by 0.1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, disodium salt (DIDS) and 79% by 1.0 mM furosemide, Na+-stimulated SO4 uptake was only inhibited 11 and 0%, respectively. K+ (20 mM), Cl (5 mM), and oxalate (0.25 mM) inhibited pH gradient-stimulated SO4 uptake (38-65%) but had no effect on Na+-stimulated SO4 uptake. Finally, at Na+ concentrations (10 mM) significantly less than that required for Na+-stimulated SO4 uptake (60-100 mM), external Na+ inhibited pH gradient-stimulated SO4 uptake, suggesting two independent effects of this cation. SO4 uptake was also inhibited by external K+ both in the presence and absence of a pH gradient. A Dixon plot of the DIDS-sensitive SO4 uptake under pH gradient conditions yielded a straight line, indicating a single site of interaction between external K+ and the SO4-OH carrier (apparent Ki = 7.2 mM). In contrast to the inhibition by external K+, internal K+ stimulated SO4 uptake. This effect was DIDS sensitive and not enhanced by valinomycin, suggesting an interaction of internal K+ with the SO4-OH exchanger independent of a K+-induced electrical potential. SO4 uptake and the effects of K+ were pH modulated with less SO4 uptake and less K+ effect at higher pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Synergistic alpha-1 and alpha-2 adrenergic stimulation of rat proximal nephron Na+/H+ exchange.

Both alpha-1 and alpha-2 adrenoceptors have been localized to the renal cortex, with the majority of binding sites on the proximal tubule. Because the major regulator of Na+ uptake into the proximal tubule is the Na+/H+ exchanger, and because alpha-1 and alpha-2 adrenoceptors stimulate it in other tissues, we tested the hypothesis that both alpha adrenoceptor subtypes can increase Na+ uptake into the proximal nephron by stimulating the Na+/H+ antiporter. Enhancement of Na+ transport by agonists was studied in isolated rat proximal tubules by determining the uptake of 22Na that was suppressible by the Na+/H+ inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA). The phorbol ester, phorbol-12-myristate-13-acetate, (0.1 microM), directly stimulated the antiporter through protein kinase C and increased EIPA-suppressible 22Na uptake 250% above control. The alpha-1 adrenoceptor agonists, cirazoline and phenylephrine, in addition to the mixed agonist, norepinephrine, maximally stimulated uptake by 226 to 232% at 1 microM concentrations. alpha-2 agonists produced a range of maximal stimulations at 1 microM from 65% with guanabenz to 251% with B-HT 933. Increases in 22Na uptake by agonists were inhibited by selective adrenergic antagonists and by EIPA. The drugs did not change the EIPA-resistant component of 22Na uptake. Inasmuch as the adrenoceptor subtypes likely stimulated Na+/H+ exchange by differing intracellular pathways impinging upon common transport steps, we examined whether simultaneous stimulation of both pathways was additive. Submaximal concentrations (5 nM each) of alpha-1 and alpha-2 adrenoceptor agonists in combination synergistically enhanced 22Na uptake to a level similar to 1 microM concentrations of adrenoceptor agonists alone or in combination.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on inactivation of anion transport in human red blood cell membrane by reversibly and irreversibly acting arginine-specific reagents.

A chromophoric derivative of phenylglyoxal, 4-hydroxy-3-nitrophenylglyoxal (HNPG), known to be highly selective for modification of arginine residues in aqueous solution is found to be a potent inhibitor of anion transport across the red cell membrane. In contrast to the action of all other arginine-specific reagents used under the experimental conditions in this laboratory, the action of HNPG on sulfate transport is completely reversible. Hence, a kinetic analysis of its inhibitory effect on SO4(2-) self-exchange could be performed. The effect of increasing chloride concentration on the inhibitory potency of HNPG is consistent with the concept that Cl- and HNPG compete for the same site on the anion transporter. The IC50 value for the inhibition of SO4(2-) exchange with HNPG is about 0.13 mM at pH 8.0 and 0.36 mM at pH 7.4, and the Hill coefficient for the interaction between the transporter and the inhibitor is near one at both pH's. HNPG is able to protect the transport system against inhibition with the (under our experimental conditions) irreversibly acting arginine specific reagent, phenylglyoxal. Partial inactivation of the transport system with phenylglyoxal lowers the maximal rates of SO4(2-) and chloride exchange but does not modify the apparent KS for the substrate anions. Reversibly acting anion transport inhibitors known to interact with the DIDS binding site like salicylate, tetrathionate, APMB, DNDS, and flufenamate are able to protect the transport system against phenylglyoxalation. Other inhibitors like phloretin and phlorizin have no effect.

Different effect of ouabain on the interferon production and action.

Ouabain, which inhibits specifically membrane-bound ATPase activity, also inhibits the establishment of the antiviral state induced by interferon. Once the antiviral state is established, ouabain is ineffective. This inhibitory effect is reversed by adding Na/K ions to the cells. On the contrary, interferon production is unaffected by the same concentrations of ouabain. It is of interest that in such interferon-yielding cells, ouabain decreases the antiviral state.

[Etiopathogenetical diagnosis and therapy of epilepsy (tentative application of some neurophysiological data)(author's transl)]. / Atiopathogenetische Diagnostik und Therapie der Epilepsie (Versuch einer Anwendung physiologischer Erkenntnisse).

A purely symptomatological classification of epilepsies can be substituted by diagnostical determination of etiopathogenetical components, which take part in the origin and evolution of the pathological process in individual cases. Usually it can be done by simple diagnostical processing. The etiopathogenetical diagnosis can also contribute to a less empirical and more rational use of therapeutical means.

Regulation of Na+ and Cl- transport and mucous gland secretion in airway epithelium.

Ussing's short-circuit technique was applied to canine airway epithelium in vitrol and a net flux of Cl- towards the airway lumen was demonstrated, with a smaller net flux of Na+ in the opposite direction. Furosemide decreased and acetylcholine, terbutaline, and histamine increased net ion transport towards the airway lumen. Associated changes in water content in the airway lumen could affect mucociliary clearance, and therefore inhibition of ion transport may play a role in disease states. To study secretions from submucosal glands in vivo, two techniques were used to identify the duct openings in the exposed canine tracheal epithelium. (a) The exposed mucosal surface was coated with powdered tantalum; accumulated secretions produced elevations under which the duct openings were located. (b) A vital dye (0.1% Neutral red) was placed on the exposed mucosal surface; the dye stained the duct openings. With these techniques, the innervation of the submucosal glands and the autonomic regulation of their secretions were studied. Micropuncture techniques were used to sample the secretions from the glands and ducts.