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Biomedical subjects

L Toro

Publications and source records attributed to L Toro.

At least 19 recordsLinked to original sources
PubMed2005

Ionic strength effect on copper biosorption by Sphaerotilus natans: equilibrium study and dynamic modelling in membrane reactor.

Biosorption of copper by Sphaerotilus natans in different conditions of ionic strength and pH was studied by performing sorption tests in batch and membrane reactors. Equilibrium batch tests evidenced the negative effect of ionic strength and the positive effect of pH on biosorption performances: the highest determined value for copper specific uptake, q, was about 60 mg/g at pH 6 and about 15 mg/g at pH 4. A competitive equilibrium model was successfully fitted to experimental data at different ionic strength levels to account for copper-sodium competition. In membrane reactor tests, experimental profiles of copper concentration in the permeate vs. time did not evidence a significant effect of ionic strength at low pH values (4 and 5). On the other hand a more remarkable effect of ionic strength on copper concentration in the permeate was observed at pH 6. Experimental profiles of continuous biosorption in the membrane reactors were successfully simulated by developing a dynamic model accounting for Cu-Na competition and for binding ability of cells fragments.

Adsorption↗
PubMed2004

An endoplasmic reticulum trafficking signal prevents surface expression of a voltage- and Ca2+-activated K+ channel splice variant.

Protein delivery to restricted plasma membrane domains is exquisitely regulated at different stages of the cell trafficking machinery. Traffic control involves the recognition of export/retention/retrieval signals in the endoplasmic reticulum (ER)/Golgi complex that will determine protein fate. A splice variant (SV), SV1, of the voltage- and Ca(2+)-activated K(+) channel alpha-subunit accumulates the channel in the ER, preventing its surface expression. We show that SV1 insert contains a nonbasic, hydrophobic retention/retrieval motif, CVLF, that does not interfere with proper folding and tetramerization of SV1. Localization of proteins in the ER by CVLF is independent of its position; originally, on the first internal loop, SV1 insert or CVLF perform equally well if placed at the middle or end of the alpha-subunit intracellular carboxyl terminus. Also, CVLF is able to restrict the traffic of an independently expressed transmembrane protein, beta 1-subunit. CVLF is present in proteins across species and in lower organisms. Thus, CVLF may have evolved to serve as a regulator of cellular traffic.

Amino Acid Motifs↗
PubMed2004

Sequential extraction of heavy metals in river sediments of an abandoned pyrite mining area: pollution detection and affinity series.

In this paper heavy metal pollution at an abandoned Italian pyrite mine has been investigated by comparing total concentrations and speciation of heavy metals (Fe, Cu, Mn, Zn, Pb and As) in a red mud sample and a river sediment. Acid digestions show that all the investigated heavy metals present larger concentrations in the sediment than in the tailing. A modified Tessier's procedure has been used to discriminate heavy metal bound to organic fraction from those originally present in the mineral sulphide matrix and to detect a possible trend of metal mobilisation from red mud to river sediment. Sequential extractions on bulk and size fractionated samples denote that sediment samples present larger percent concentrations of the investigated heavy metals in the first extractive steps (I-IV) especially in lower dimension size fractionated samples suggesting that heavy metals in the sediment are significantly bound by superficial adsorption mechanisms.

Adsorption↗
PubMed2004

Differential expression of alpha 1 and beta subunits of voltage dependent Ca2+ channel at the neuromuscular junction of normal and P/Q Ca2+ channel knockout mouse.

Voltage-dependent calcium channels (VDCC) have a key role in neuronal function transforming the voltage signals into intracellular calcium signals. They are composed of the pore-forming alpha(1) and the regulatory alpha(2)delta, gamma and beta subunits. Molecular and functional studies have revealed which alpha(1) subunit gene product is the molecular constituent of each class of native calcium channel (L, N, P/Q, R and T type). Electrophysiological and immunocytochemical studies have suggested that at adult mouse motor nerve terminal (MNT) only P/Q type channels, formed by alpha(1A) subunit, mediate evoked transmitter release. The generation of alpha(1A)-null mutant mice offers an opportunity to study the expression and localization of calcium channels at a synapse with complete loss of P/Q calcium channel. We have investigated the expression and localization of VDCCs alpha(1) and beta subunits at the wild type (WT) and knockout (KO) mouse neuromuscular junction (NMJ) using fluorescence immunocytochemistry. The alpha(1A) subunit was observed only at WT NMJ and was absent at denervated muscles and at KO NMJ. The subunits alpha(1B), alpha(1D) and alpha(1E) were also present at WT NMJ and they were over- expressed at KO NMJ suggesting a compensatory expression due to the lack of the alpha(1A). On the other hand, the beta(1b), beta(2a) and beta(4) were present at the same levels in both genotypes. The presence of other types of VDCC at WT NMJ indicate that they may play other roles in the signaling process which have not been elucidated and also shows that other types of VDCC are able to substitute the alpha(1A) subunit, P/Q channel under certain pathological conditions.

Animals↗
PubMed2003

Altered functional coupling of coronary K+ channels in diabetic dyslipidemic pigs is prevented by exercise.

Chronic hyperglycemia and hypercholesterolemia have been shown to alter ionic currents in vascular smooth muscle. We tested the hypothesis that the combined effect of hyperglycemia and hyperlipidemia (diabetic dyslipidemia) would increase the Ca2+-sensitive K+ (KCa) current as a compensatory response to an increase in intracellular Ca2+ concentration. We also hypothesized that exercise training would prevent this elevation in KCa current. Miniature Yucatan swine were randomly assigned to five groups: control, standard pig chow (C, n = 6); hyperlipidemic, high-fat pig chow (H, n = 5); diabetic, standard pig chow (D, n = 7); diabetic, high-fat pig chow ("diabetic dyslipidemic," DD, n = 12); and exercise-trained DD (DDX, n = 9). High-fat chow consisted of standard minipig chow supplemented with cholesterol (2%) and coconut oil. Increased coronary vasoconstriction assessed in vivo and in vitro in DD was prevented by exercise. Patch-clamp experiments performed on right coronary artery smooth muscle cells resulted in greater K+ current densities in the H, D, and DD groups vs. the DDX group between -10 and 40 mV. In fura 2-loaded cells, current activated by caffeine-induced Ca2+ release was greater in H, D, and DD compared with C and DDX (P < 0.05), whereas intracellular Ca2+ concentration was not different across groups. Finally, there were no differences in the KCa or Kv channel protein content between groups. These data indicate that hyperglycemia, hyperlipidemia, and diabetic dyslipidemia lead to elevated whole cell K+ current and increased functional coupling of KCa and Ca2+ release. Endurance exercise prevented increased coupling of Ca2+ release to KCa channel activation in diabetic dyslipidemia.

Animals↗
PubMed2003

Metal speciation and pH effect on Pb, Cu, Zn and Cd biosorption onto Sphaerotilus natans: Langmuir-type empirical model.

Biosorption data of lead, copper, zinc and cadmium onto Sphaerotilus natans at different equilibrium pH (3-5 units) were here reported and analysed. Experimental results outlined the positive effect of pH increase on pollutant uptake and also the biomass affinity series (Pb > Cu > Zn > Cd) reflecting the hydrolytic properties of metals. An original empirical model was proposed to represent the effect of pH on heavy metal biosorption inserting q(max) vs. pH empirical functions into the classical Langmuir isotherm.

Adsorption↗
PubMed2002

External pore collapse as an inactivation mechanism for Kv4.3 K+ channels.

Kv4 channels are thought to lack a C-type inactivation mechanism (collapse of the external pore) and to inactivate as a result of a concerted action of cytoplasmic regions of the channel. To investigate whether Kv4 channels have outer pore conformational changes during the inactivation process, the inactivation properties of Kv4.3 were characterized in 0 mM and in 2 mM external K+ in whole-cell voltage-clamp experiments. Removal of external K+ increased the inactivation rates and favored cumulative inactivation by repetitive stimulation. The reduction in current amplitude during repetitive stimulation and the faster inactivation rates in 0 mM external K+ were not due to changes in the voltage dependence of channel opening or to internal K+ depletion. The extent of the collapse of the K+ conductance upon removal of external K+ was more pronounced in NMG+-than in Na+-containing solutions. The reduction in the current amplitude during cumulative inactivation by repetitive stimulation is not associated with kinetic changes, suggesting that it is due to a diminished number of functional channels with unchanged gating properties. These observations meet the criteria for a typical C-type inactivation, as removal of external K+ destabilizes the conducting state, leading to the collapse of the pore. A tentative model is presented, in which K+ bound to high-affinity K+-binding sites in the selectivity filter destabilizes an outer neighboring K+ modulatory site that is saturated at approximately 2 mM external K+. We conclude that Kv4 channels have a C-type inactivation mechanism and that previously reported alterations in the inactivation rates after N- and C- termini mutagenesis may arise from secondary changes in the electrostatic interactions between K+-binding sites in the selectivity filter and the neighboring K+-modulatory site, that would result in changes in its K+ occupancy.

Cell Line↗
PubMed2001

Slotoxin, alphaKTx1.11, a new scorpion peptide blocker of MaxiK channels that differentiates between alpha and alpha+beta (beta1 or beta4) complexes.

A novel peptide from Centruroides noxius Hoffmann scorpion venom was isolated and sequenced. The 37 amino acid peptide belongs to the charybdotoxin sub-family (alphaKTx1) and was numbered member 11. alphaKTx1.11 has 75% sequence identity with iberiotoxin and 54% with charybdotoxin. alphaKTx1.11 revealed specificity for mammalian MaxiK channels (hSlo), thus, was named slotoxin. Slotoxin blocks the MaxiK pore-forming alpha subunit reversibly (K(d)=1.5 nM). Slotoxin association with alpha+beta (beta1 or beta4) channels was approximately 10 times slower than iberiotoxin and charybdotoxin, leading to a lack of effect on alpha+beta4 when tested at 100 nM for 5 min. Thus, slotoxin is a better tool to distinguish MaxiK alpha+beta complexes.

Amino Acid Sequence↗
PubMed2001

Remodeling of Kv4.3 potassium channel gene expression under the control of sex hormones.

Kv4.3 channels are important molecular components of transient K(+) currents (Ito currents) in brain and heart. They are involved in setting the frequency of neuronal firing and heart pacing. Altered Kv4.3 channel expression has been demonstrated under pathological conditions like heart failure indicating their critical role in heart function. Thyroid hormone studies suggest that their expression in the heart may be hormonally regulated. To explore the possibility that sex hormones control Kv4.3 expression, we investigated whether its expression changes in the pregnant uterus. This organ represents a unique model to study Ito currents, because it possesses this type of K(+) current and undergoes dramatic changes in function and excitability during pregnancy. We cloned Kv4.3 channel from myometrium and found that its protein and transcript expression is greatly diminished during pregnancy. Experiments in ovariectomized rats demonstrate that estrogen is one mechanism responsible for the dramatic reduction in Kv4.3 expression and function prior to parturition. Furthermore, the reduction of plasma membrane Kv4.3 protein is accompanied by a perinuclear localization suggesting that cell trafficking is also controlled by sex hormones. Thus, estrogen remodels the expression of Kv4.3 in myometrium by directly diminishing its transcription and, indirectly, by altering Kv4.3 delivery to the plasma membrane.

Animals↗
PubMed2001

A novel MaxiK splice variant exhibits dominant-negative properties for surface expression.

We identified a novel MaxiK alpha subunit splice variant (SV1) from rat myometrium that is also present in brain. SV1 has a 33-amino acid insert in the S1 transmembrane domain that does not alter S1 overall hydrophobicity, but makes the S0-S1 linker longer. SV1 was transfected in HEK293T cells and studied using immunocytochemistry and electrophysiology. In non-permeabilized cells, N-terminal c-Myc- or C-terminal green fluorescent protein-tagged SV1 displayed no surface labeling or currents. The lack of SV1 functional expression was due to endoplasmic reticulum (ER) retention as determined by colabeling experiments with a specific ER marker. To explore the functional role of SV1, we coexpressed SV1 with the alpha (human SLO) and beta1 (KCNMB1) subunits of the MaxiK channel. Coexpression of SV1 inhibited surface expression of alpha and beta1 subunits approximately 80% by trapping them in the ER. This inhibition seems to be specific for MaxiK channel subunits since SV1 was unable to prevent surface expression of the Kv4.3 channel or to interact with green fluorescent protein. These results indicate a dominant-negative role of SV1 in MaxiK channel expression. Moreover, they reveal down-regulation by splice variants as a new mechanism that may contribute to the diverse levels of MaxiK channel expression in non-excitable and excitable cells.

Alternative Splicing↗
PubMed2001

Decreased expression of voltage- and Ca(2+)-activated K(+) channels in coronary smooth muscle during aging.

Aging is the main risk factor for coronary artery disease. One characteristic of aging coronary arteries is their enhanced contractile responses to endothelial vasoconstricting factors, which increase the risk of coronary vasospasm in older people. Because large-conductance voltage- and Ca(2+)-activated K(+) channels (MaxiK) are key regulators of vascular tone, we explored the possibility that this class of channels is diminished with increasing age. Using site-directed antibodies recognizing the pore-forming alpha subunit and electrophysiological methods, we demonstrate that the number of MaxiK channels is dramatically diminished in aged coronary arteries from old F344 rats. Channel density was reduced from 52+/-9 channels/pF (3 months old) to 18+/-5 channels/pF (25 to 30 months old), which represents a 65% reduction in the older population. Pixel intensity of Western blots was also diminished by approximately 50%. Moreover, the age-related decrease in the channel protein expression was also evident in humans, which showed approximately 80% reduction in 61- to 70-year-old subjects compared with 3- to 18-year-old youngsters and approximately 45% reduction compared with 19- to 56-year-old adults. In agreement with a reduction of MaxiK channel numbers in aging coronary arteries, old coronary arteries from F344 rats contract less effectively ( approximately 70% reduction) than young coronary arteries when exposed to the MaxiK channel blocker iberiotoxin. The contraction studies indicate that under physiological conditions, MaxiK channels are tonically active, serving as a hyperpolarizing force that opposes contraction. Thus, reduced expression of MaxiK channels in aged coronary arteries would lead to a decreased vasodilating capacity and increased risk of coronary spasm and myocardial ischemia in older people.

Adolescent↗
PubMed2001

MaxiK channel-mediated relaxation of guinea-pig aorta following stimulation of IP receptor with beraprost via cyclic AMP-dependent and -independent mechanisms.

The present study was aimed to elucidate the cellular pathway(s) controlling vascular relaxation triggered by stimulation of prostaglandin I2 (PGI2, IP) receptor with a stable PGI2 analog, beraprost. Beraprost caused a concentration-dependent relaxation in de-endothelialized guinea-pig aorta contracted with prostaglandin F2alpha (PGF2alpha). Beraprost-induced relaxation was almost abolished in high-KCl-contracted tissue, indicating a major role of K+ conductances. In contrast to other PGI2 analogs (e.g. cicaprost and iloprost), beraprost-induced relaxation was practically abolished by a selective voltage and Ca2+-activated K+ (MaxiK, BK) channel blocker Iberiotoxin (10(-7) M) or by tetraethylammonium (2 x 10(-3) M). The relaxation induced by beraprost was not significantly affected by other K+ channel blockers glibenclamide (10(-6) M) or Ba2+ (10(-5) M), but was slightly attenuated by 4-aminopyridine (10(-4) M). Beraprost increased intracellular cyclic AMP levels, suggesting a role for cyclic AMP-dependent pathways. A selective inhibitor of cyclic AMP-specific phosphodiesterase, RO-20-1724 (10(-4) M), significantly potentiated beraprost-induced relaxation. Iberiotoxin (10(-7) M) completely counteracted this potentiation. Moreover, tension decrement due to forskolin (3 x 10(-7) M) or 8-bromo-cyclic AMP (10(-2) M) was thoroughly restored by Iberiotoxin (10(-7) M), confirming a role for a cyclic AMP-dependent mechanism. However, SQ 22,536 (10(-4) M), an adenylyl cyclase inhibitor, did not affect beraprost-induced relaxation though it almost totally inhibited the elevation of cyclic AMP contents induced by beraprost, suggesting the existence of an additional mechanism that is cyclic AMP-independent. Moreover, cholera toxin (CTX, 1 microg/ml for 6 h), which activates the stimulatory G protein of adenylyl cyclase (Gs), significantly suppressed PGF2alpha-induced contraction both in the absence and presence of SQ 22,536 (10(-4) M). Iberiotoxin (10(-7) M) was also capable of restoring the relaxation induced by CTX. These findings suggest that MaxiK channel plays a primary role in mediating smooth muscle relaxation following stimulation of IP receptor with beraprost in guinea-pig aorta. Both cyclic AMP-dependent and -independent pathways contribute to the MaxiK channel-mediated relaxation following IP receptor stimulation in this vascular tissue. Direct regulation of MaxiK channels by Gs may partly account for the cyclic AMP-independent relaxant mechanism.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2000

Apical sorting of a voltage- and Ca2+-activated K+ channel alpha -subunit in Madin-Darby canine kidney cells is independent of N-glycosylation.

The voltage- and Ca(2+)-activated K(+) (K(V,Ca)) channel is expressed in a variety of polarized epithelial cells seemingly displaying a tissue-dependent apical-to-basolateral regionalization, as revealed by electrophysiology. Using domain-specific biotinylation and immunofluorescence we show that the human channel K(V,Ca) alpha-subunit (human Slowpoke channel, hSlo) is predominantly found in the apical plasma membrane domain of permanently transfected Madin-Darby canine kidney cells. Both the wild-type and a mutant hSlo protein lacking its only potential N-glycosylation site were efficiently transported to the cell surface and concentrated in the apical domain even when they were overexpressed to levels 200- to 300-fold higher than the density of intrinsic Slo channels. Furthermore, tunicamycin treatment did not prevent apical segregation of hSlo, indicating that endogenous glycosylated proteins (e.g., K(V,Ca) beta-subunits) were not required. hSlo seems to display properties for lipid-raft targeting, as judged by its buoyant distribution in sucrose gradients after extraction with either detergent or sodium carbonate. The evidence indicates that the hSlo protein possesses intrinsic information for transport to the apical cell surface through a mechanism that may involve association with lipid rafts and that is independent of glycosylation of the channel itself or an associated protein. Thus, this particular polytopic model protein shows that glycosylation-independent apical pathways exist for endogenous membrane proteins in Madin-Darby canine kidney cells.

Animals↗
PubMed2000

A neuronal beta subunit (KCNMB4) makes the large conductance, voltage- and Ca2+-activated K+ channel resistant to charybdotoxin and iberiotoxin.

Large conductance voltage and Ca(2+)-activated K(+) (MaxiK) channels couple intracellular Ca(2+) with cellular excitability. They are composed of a pore-forming alpha subunit and modulatory beta subunits. The pore blockers charybdotoxin (CTx) and iberiotoxin (IbTx), at nanomolar concentrations, have been invaluable in unraveling MaxiK channel physiological role in vertebrates. However in mammalian brain, CTx-insensitive MaxiK channels have been described [Reinhart, P. H., Chung, S. & Levitan, I. B. (1989) Neuron 2, 1031-1041], but their molecular basis is unknown. Here we report a human MaxiK channel beta-subunit (beta4), highly expressed in brain, which renders the MaxiK channel alpha-subunit resistant to nanomolar concentrations of CTx and IbTx. The resistance of MaxiK channel to toxin block, a phenotype conferred by the beta4 extracellular loop, results from a dramatic ( approximately 1,000 fold) slowdown of the toxin association. However once bound, the toxin block is apparently irreversible. Thus, unusually high toxin concentrations and long exposure times are necessary to determine the role of "CTx/IbTx-insensitive" MaxiK channels formed by alpha + beta4 subunits.

Amino Acid Sequence↗
PubMed1999

Hormonal control of protein expression and mRNA levels of the MaxiK channel alpha subunit in myometrium.

Large conductance voltage-dependent and Ca(2+)-modulated K(+) channels play a crucial role in myometrium contractility. Western blots and immunocytochemistry of rat uterine sections or isolated cells show that MaxiK channel protein signals drastically decrease towards the end of pregnancy. Consistent with a transcriptional regulation of channel expression, mRNA levels quantified with the ribonuclease protection assay correlated well with MaxiK protein levels. As a control, Na(+)/K(+)-ATPase protein and RNA levels do not significantly change at different stages of pregnancy. The low numbers of MaxiK channels at the end of pregnancy may facilitate uterine contraction needed for parturition.

Animals↗
PubMed1999

Role of MaxiK channels in vasoactive intestinal peptide-induced relaxation of rat mesenteric artery.

We investigated the functional relevance of large conductance voltage-dependent and Ca(2+)-sensitive K(+)(MaxiK) channels in vasoactive intestinal peptide (VIP)-induced relaxation of rat mesenteric artery. VIP, which is known to increase cAMP levels, produced a concentration-dependent relaxation in endothelium-denuded arteries. Iberiotoxin, a MaxiK channel blocker, greatly diminished the VIP-induced relaxation. In a similar manner, a significant portion of the relaxant response to dibutyryl-cAMP (DBcAMP), a membrane-permeable analog of cAMP, was inhibited by iberiotoxin. These results suggest that activation of MaxiK channels significantly contributes to the relaxant response of rat mesenteric artery to VIP, possibly via cAMP-mediated pathways.

Animals↗