(1/4132) Modulation of slow inactivation in human cardiac Kv1.5 channels by extra- and intracellular permeant cations.

1. The properties and regulation of slow inactivation by intracellular and extracellular cations in the human heart K+ channel hKv1.5 have been investigated. Extensive NH2- and COOH-terminal deletions outside the central core of transmembrane domains did not affect the degree of inactivation. 2. The voltage dependence of steady-state inactivation curves of hKv1.5 channels was unchanged in Rb+ and Cs+, compared with K+, but biexponential inactivation over 10 s was reduced from approximately 100 % of peak current in Na+ to approximately 65 % in K+, approximately 50 % in Rb+ and approximately 30 % in Cs+. This occurred as a result of a decrease in both fast and slow components of inactivation, with little change in inactivation time constants. 3. Changes in extracellular cation species and concentration (5-300 mM) had only small effects on the rates of inactivation and recovery from inactivation (tau recovery approximately 1 s). Mutation of residues at a putative regulatory site at R487 in the outer pore mouth did not affect slow inactivation or recovery from inactivation of hKv1.5, although sensitivity to extracellular TEA was conferred. 4. Symmetrical reduction of both intra- and extracellular cation concentrations accelerated and augmented both components of inactivation of K+ (Kd = 34.7 mM) and Cs+ (Kd = 20.5 mM) currents. These effects could be quantitatively accounted for by unilateral reduction of intracellular K+ (K+i) (Kd = 43.4 mM) or Cs+i with constant 135 mM external ion concentrations. 5. We conclude that inactivation and recovery from inactivation in hKv1.5 were not typically C-type in nature. However, the ion species dependence of inactivation was still closely coupled to ion permeation through the pore. Intracellular ion modulatory actions were more potent than extracellular actions, although still of relatively low affinity. These results suggest the presence of ion binding sites capable of regulating inactivation located on both intracellular and extracellular sides of the pore selectivity filter.  (+info)

(2/4132) Genetic and biochemical determinants of abnormal monovalent ion transport in primary hypertension.

Data obtained during the last two decades show that spontaneously hypertensive rats, an acceptable experimental model of primary human hypertension, possess increased activity of both ubiquitous and renal cell-specific isoforms of the Na+/H+ exchanger (NHE) and Na+-K+-2Cl- cotransporter. Abnormalities of these ion transporters have been found in patients suffering from essential hypertension. Recent genetic studies demonstrate that genes encoding the beta- and gamma-subunits of ENaC, a renal cell-specific isoform of the Na+-K+-2Cl- cotransporter, and alpha3-, alpha1-, and beta2-subunits of the Na+-K+ pump are localized within quantitative trait loci (QTL) for elevated blood pressure as well as for enhanced heart-to-body weight ratio, proteinuria, phosphate excretion, and stroke latency. On the basis of the homology of genome maps, several other genes encoding these transporters, as well as the Na+/H+ exchanger and Na+-K+-2Cl- cotransporter, can be predicted in QTL related to the pathogenesis of hypertension. However, despite their location within QTL, analysis of cDNA structure did not reveal any mutation in the coding region of the above-listed transporters in primary hypertension, with the exception of G276L substitution in the alpha1-Na+-K+ pump from Dahl salt-sensitive rats and a higher occurrence of T594M mutation of beta-ENaC in the black population with essential hypertension. These results suggest that, in contrast to Mendelian forms of hypertension, the altered activity of monovalent ion transporters in primary hypertension is caused by abnormalities of systems involved in the regulation of their expression and/or function. Further analysis of QTL in F2 hybrids of normotensive and hypertensive rats and in affected sibling pairs will allow mapping of genes causing abnormalities of these regulatory pathways.  (+info)

(3/4132) Enteropathogenic E. coli attenuates secretagogue-induced net intestinal ion transport but not Cl- secretion.

Enteric bacterial pathogens often increase intestinal Cl- secretion. Enteropathogenic Escherichia coli (EPEC) does not stimulate active ion secretion. In fact, EPEC infection decreases net ion transport in response to classic secretagogues. This has been presumed to reflect diminished Cl- secretion. The aim of this study was to investigate the influence of EPEC infection on specific intestinal epithelial ion transport processes. T84 cell monolayers infected with EPEC were used for these studies. EPEC infection significantly decreased short-circuit current (Isc) in response to carbachol and forskolin, yet 125I efflux studies revealed no difference in Cl- channel activity. There was also no alteration in basolateral K+ channel or Na+-K+-2Cl- cotransport activity. Furthermore, net 36Cl- flux was not decreased by EPEC. No alterations in either K+ or Na+ transport could be demonstrated. Instead, removal of basolateral bicarbonate from uninfected monolayers yielded an Isc response approximating that observed with EPEC infection, whereas bicarbonate removal from EPEC-infected monolayers further diminished Isc. These studies suggest that the reduction in stimulated Isc is not secondary to diminished Cl- secretion. Alternatively, bicarbonate-dependent transport processes appear to be perturbed.  (+info)

(4/4132) Nitric oxide-mediated regulation of transepithelial sodium and chloride transport in murine nasal epithelium.

Transepithelial ion transport is regulated by a variety of cellular factors. In light of recent evidence that nitric oxide (NO) production is decreased in cystic fibrosis airways, we examined the role of NO in regulating sodium and chloride transport in murine nasal epithelium. Acute intervention with the inducible NO synthase (iNOS)-selective inhibitor S-methylisothiourea resulted in an increase of amiloride-sensitive sodium absorption observed as a hyperpolarization of nasal transepithelial potential difference. Inhibition of iNOS expression with dexamethasone also hyperpolarized transepithelial potential difference, but only a portion of this increase proved to be amiloride sensitive. Chloride secretion was significantly inhibited in C57BL/6J mice by the addition of both S-methylisothiourea and dexamethasone. Mice lacking iNOS expression [NOS2(-/-)] also had a decreased chloride-secretory response compared with control mice. These data suggest that constitutive NO production likely plays some role in the downregulation of sodium absorption and leads to an increase in transepithelial chloride secretion.  (+info)

(5/4132) Regional electrophysiological effects of hypokalaemia, hypomagnesaemia and hyponatraemia in isolated rabbit hearts in normal and ischaemic conditions.

OBJECTIVE: The aims of this study were to establish an isolated working heart model for electrophysiological recordings from the epicardium and endocardium and to examine regional effects of changes in ion concentrations in normal and ischaemic conditions. METHODS: Monophasic action potential duration (MAPD90), effective refractory period (ERP) and conduction delay were measured simultaneously in the epicardium and endocardium of rabbit hearts paced at 3.3 Hz, subjected to 30 min of regional ischaemia and 15 min of reperfusion. The hearts were exposed before and throughout ischaemia and reperfusion to hypokalaemia (K+ = 2 mM), hypomagnesaemia (Mg2+ = 0.5 mM) or hyponatraemia (Na+ = 110 mM). RESULTS: In the control hearts, no regional electrophysiological differences were seen before ischaemia, but ischaemia-induced MAPD90 shortening and postrepolarisation refractoriness were greater in the epicardium than in the endocardium and conduction delay increased only in the epicardium. Hypokalaemia shortened ERP in the epicardium (but not endocardium) and increased conduction delay in all areas before ischaemia, but it had no effects during ischaemia. During reperfusion hypokalaemia increased the incidence of recurrent tachyarrhythmias. Hypomagnesaemia had no effect before ischaemia, increased epicardial (but not endocardial) MAPD90 shortening during ischaemia, although it had no pro-arrhythmic action. Hyponatraemia increased conduction delay in all areas before ischaemia and produced asystole or severe bradycardia in all hearts. During ischaemia, hyponatraemia decreased ERP shortening and inducibility of arrhythmias in the epicardium (but not endocardium). CONCLUSIONS: We conclude that the more pronounced effect of ischaemia upon the epicardium than the endocardium can be explained by the contact of the endocardium with intracavitary perfusate. We also conclude that changes in ion concentrations may have differential regional electrical effects in normal or ischaemic conditions.  (+info)

(6/4132) Temperature sensitivity of catecholamine secretion and ion fluxes in bovine adrenal chromaffin cells.

The effects of temperature on ion fluxes and catecholamine secretion that are mediated by nicotinic acetylcholine receptors (nAChRs), voltage-sensitive calcium channels (VSCCs), and voltage-sensitive sodium channels (VSSCs) were investigated using bovine adrenal chromaffin cells. When the chromaffin cells were stimulated with DMPP, a nicotinic cholinergic agonist, or 50 mM K+, the intracellular calcium ([Ca2+]i) elevation reached a peak and decreased more slowly at lower temperatures. The DMPP-induced responses were more sensitive to temperature changes compared to high K+-induced ones. In the measurement of intracellular sodium concentrations ([Na+]i), it was found that nicotinic stimulation required a longer time to attain the maximal level of [Na+]i at lower temperatures. In addition, the VSSCs-mediated [Na+]i increase evoked by veratridine was also reduced as the temperature decreased. The measurement of [3H]norepinephrine (NE) secretion showed that the secretion within the first 3 min evoked by DMPP or high K+ was greatest at 37 degrees C. However, at 25 degrees C, the secretion evoked by DMPP, but not that by the 50 mM K+, was greater after 10 min of stimulation. This data suggest that temperature differentially affects the activity of nAChRs, VSCCs, and VSSCs, resulting in differential [Na+]i and [Ca2+]i elevation, and in the [3H]NE secretion by adrenal chromaffin cells.  (+info)

(7/4132) Release of copper ions from the familial amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutants.

Point mutations of Cu,Zn-superoxide dismutase (SOD) have been linked to familial amyotrophic lateral sclerosis (FALS). We reported that the Swedish FALS Cu,Zn-SOD mutant, D90A, exhibited an enhanced hydroxyl radical-generating activity, while its dismutation activity was identical to that of the wild-type enzyme (Kim et al. 1998a; 1998b). Transgenic mice that express a mutant Cu,Zn-SOD, Gly93 --> Ala (G93A), have been shown to develop amyotrophic lateral sclerosis (ALS) symptoms. We cloned the cDNA for the FALS G93A mutant, overexpressed the protein in E. coli cells, purified the protein, and studied its enzymic activities. Our results showed that the G93A, the D90A, and the wild-type enzymes have identical dismutation activity. However, the hydroxyl radical-generating activity of the G93A mutant was enhanced relative to those of the D90A and the wild-type enzyme (wild-type < D90A < G93A). These higher free radical-generating activities of mutants facilitated the release of copper ions from their own molecules (wild-type < D90A < G93A). The released copper ions can enhance the Fenton-like reaction to produce hydroxyl radicals and play a major role in the oxidative damage of macromolecules. Thus, the FALS symptoms may be associated with the enhancements in both the free radical-generating activity and the releasing of copper ions from the mutant enzyme.  (+info)

(8/4132) Determination of tin, vanadium, iron, and molybdenum in various matrices by atomic absorption spectrometry using a simultaneous liquid-liquid extraction procedure.

An atomic-absorption spectrometric method is described for the determination of tin, vanadium, iron, and molybdenum in two certified reference materials, food samples, and petroleum crude. After treatment with acids, these elements are separated from matrix elements by simultaneous solvent extraction of 5,5'-methylenedisalicylohydroxamic acid complexes from HCl/NaClO4 solution into an isobutyl methyl ketone/tributyl phosphate solution. The detection limits range from 0.018 to 0.19 microg/mL (n = 3), and the relative standard deviations do not exceed 2.0% at levels of 0.5, 0.6, 2.0, and 7.0 microg/mL of Fe, Mo, V, and Sn, respectively. The method is selective and suffers only from interference by Zr(IV), Ti(IV), Th(IV), W(VI), PO4(3-), and F-.  (+info)