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L Vaca

Publications and source records attributed to L Vaca.

34 records · Page 2Linked to original sources
PubMed1995

IP3-activated Ca2+ channels in the plasma membrane of cultured vascular endothelial cells.

Although it is clear that D-myo-inositol 1,4,5-trisphosphate (IP3) plays an important role in the activation of Ca2+ influx, the mechanisms by which this occurs remain controversial. In an attempt to determine the role of IP3 in the activation of Ca2+ influx, patch-clamp single-channel experiments in the cell-attached, inside-out, and outside-out configurations were performed on cultured bovine aortic endothelial cells (BAEC). The results presented indicate that both IP3 and intracellular Ca2+ can modulate the activity of a Ca(2+)-selective channel found in the plasma membrane of these cells. Addition of 10 microM IP3 increased channel open probability (P(o)) from a control value of 0.12 +/- 0.05 to 0.7 +/- 0.13 at a constant intracellular Ca2+ of 1 nM in excised inside-out patches. D-Myo-inositol 1,3,4,5-tetrakisphosphate at 50 microM was ineffective in altering channel P(o). Channel activity declined after approximately 2 min in the continuous presence of IP3. Three to four minutes after addition of IP3, channel P(o) was reduced from 0.7 +/- 0.2 to 0.2 +/- 0.1, indicating that an additional regulator might be required to maintain channel activity in excised patches. The channel was reversibly blocked by application of 1 microgram/ml heparin to the intracellular side of inside-out patches. This Ca(2+)-selective channel is indistinguishable from the depletion-activated Ca2+ channel we have previously described in BAEC.

Animals↗
PubMed1995

Ins(1,4,5)P3 activates Drosophila cation channel Trpl in recombinant baculovirus-infected Sf9 insect cells.

The trp-like (trpl) gene product (Trpl) is thought to form a nonselective cation channel important for signal transduction in Drosophila photoreceptor cells. This channel may be the insect homologue of mammalian channels involved in Ca2+ signal transduction. To determine the mechanism of receptor-mediated activation of Trpl, whole cell membrane currents were examined in Sf9 insect cells after infection with recombinant baculovirus. Stimulation by bradykinin increased whole cell Trpl currents three- to fivefold. Similar activation of Trpl was observed by inclusion of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in the pipette solution during whole cell recordings. These currents were 1) not seen in noninfected cells or in cells expressing only the B2 receptor, 2) mimicked by D-myo-inositol 2,4,5-trisphosphate, and 3-deoxy-3-fluoro-D-myo-inositol 1,4,5-trisphosphate, 3) not seen with D-myo-inositol 1,4-bisphosphate or D-myo-inositol 1,3,4,5-tetrakisphosphate, and 4) blocked by heparin, but not by de-N-sulfated heparin. In contrast, Trpl currents were unaffected by thapsigargin. These results demonstrate that the Trpl cation channel is activated by Ins(1,4,5)P3 in a heparin-sensitive fashion. Regulation of channel activity by Ins(1,4,5)P3 may occur by a number of mechanisms, including direct binding of Ins(1,4,5)P3 to the Trpl channel or direct physical interaction between the Ins(1,4,5)P3 receptor/Ca(2+)-release channel of the endoplasmic reticulum and the Trpl protein.

Animals↗
PubMed1994

Appearance of a novel Ca2+ influx pathway in Sf9 insect cells following expression of the transient receptor potential-like (trpl) protein of Drosophila.

Activation of phospholipase C, elevation of free cytosolic Ca2+ concentration ([Ca2+]i) and stimulation of Ca2+ influx have been implicated in Drosophila phototransduction. Electrophysiological studies suggest that trp and trpl proteins may be important for the light-activated Ca2+ current found in Drosophila photoreceptor cells. Although these proteins exhibit homologies to voltage-gated Ca2+ and Na+ channels, their actual function in insect cells and their relation to proteins involved in mammalian cell Ca2+ signaling remains unknown. In the present study, [Ca2+]i was examined in fura-2-loaded Sf9 insect cells infected with recombinant baculovirus containing cDNA for the trpl protein. Ca2+ influx was examined by use of Ba2+, a Ca2+ surrogate that is not a substrate for Ca(2+)-pumps or carriers and by measurement of whole-cell membrane currents. The results suggest that expression of trpl is associated with appearance of a Ca2+ permeable, non-selective cation channel formed by the trpl protein.

Animals↗
PubMed1994

Blockade of the inward rectifier potassium current by the Ca(2+)-ATPase inhibitor 2',5'-di(tert-butyl)-1,4-benzohydroquinone (BHQ).

1. We have investigated the effect of 2',5'-di (tert-butyl)-1,4-benzohydroquinone (BHQ) and thapsigargin, inhibitors of the intracellular Ca(2+)-ATPase, on ionic currents in rat basophilic leukaemia (RBL-2H3) cells under whole cell voltage clamp. 2. The whole cell current was inwardly rectifying and reversed at -35 +/- 6 mV (n = 16). The conductance of the inward current increased as the concentration of extracellular K+ was raised from 2.7 to 5.4, 10.8 and 21.6 mM. BaCl2 (100 microM) reduced the current to a small linear component and shifted the reversal potential to -4 +/- 3 mV (n = 6). A concentration of 50 microM BaCl2 produced 45 +/- 10% (n = 4) blockade of the inward current. 3. BHQ and thapsigargin were examined for their effects on the inwardly rectifying current. A maximal blockade of inward current was obtained within 6 min after perfusion with 10 microM BHQ. The small current remaining after blockade with BHQ had a linear voltage-dependence and reversed direction at -6 +/- 9 mV (n = 6). Thapsigargin (up to 3 microM) was without effect on the inward rectifier. 4. In contrast to the blockade of the inward rectifier produced by BaCl2 which was predominantly on the steady state current, particularly at the very hyperpolarized holding potentials (-120 mV), blockade by BHQ was equally strong on the instantaneous as well as the steady state current. 5. Blockade of the inward rectifier by BHQ may cause depolarization of the cell which will affect Ca2+ influx during investigations with BHQ. Thapsigargin does not block the inward rectifier and will not inhibit Ca2+ influx in this way.

Animals↗
PubMed1994

Depletion of intracellular Ca2+ stores activates a Ca(2+)-selective channel in vascular endothelium.

The present study was designed to identify the channel responsible for Ca2+ influx after depletion of intracellular Ca2+ stores. Different maneuvers that deplete intracellular Ca2+ stores activated a Ca(2+)-selective channel. Superfusion of single bovine aortic endothelial cells with 50 nmol/l bradykinin, 10 mumol/l ATP, or 10 mumol/l 2,5-di(tert-butyl)-1,4-benzohydroquinone produced activation of channels of the same amplitude in cell-attached patches. Channel activity declined within the first minute after patch excision. The channel showed strong inward rectification and a reversal potential of 0 mV in symmetrical sodium sulfate (Na2SO4) solution. Under these conditions, the conductance was 5 pS in the inward direction. Addition of 10 mmol/l Ca2+ to the extracellular solution shifted the reversal potential to +30 +/- 5 mV, and the conductance for inward current was 11 pS. The reversal potential was used to calculate an ion permeability ratio of Ca2+/Na+ > 10:1.

Adenosine Triphosphate↗
PubMed1994

Activation of recombinant trp by thapsigargin in Sf9 insect cells.

The mammalian protein responsible for Ca2+ release-activated current (Icrac) may be homologous to the Drosophila protein designated trp. Thus the activity of trp, and another Drosophila protein designated trp-like or trpl, may be linked to depletion of the internal Ca2+ store via the so-called capacitative Ca2+ entry mechanism. To test this hypothesis, the effect of thapsigargin, a selective inhibitor of the endoplasmic reticulum Ca2+ pump, on trp- and trpl-induced whole cell membrane current was determined using the baculovirus Sf9 insect cell expression system. The results demonstrate that trp and trpl form Ca(2+)-permeable cation channels. The trpl encodes a nonselective cation channel that is constitutively active under basal nonstimulated conditions and is unaffected by thapsigargin, whereas trp is more selective for Ca2+ than Na+ and is activated by depletion of the internal Ca2+ store. Although evaluation of cation selectivity suggests that trp is not identical to the channel responsible for Icrac, these channels must share some structural feature(s) since both are activated by thapsigargin. A unique proline-rich region in the COOH-terminal tail of trp, which is absent in trpl, may be necessary for capacitative Ca2+ entry.

Animals↗
PubMed1993

Blockade of a KCa channel with synthetic peptides from noxiustoxin: a K+ channel blocker.

Using the outside-out configuration of the patch-clamp method, we studied the effect of several synthetic peptides corresponding to various segments from the N-terminal region of noxiustoxin (NTX) on single Ca(2+)-activated K+ (KCa) channels of small conductance obtained from cultured bovine aortic endothelial cells. These peptides induced diverse degrees of fast blockade in the endothelial KCa channel. The most effective blockers were the peptides NTX1-39 (IC50 = 0.5 microM) and NTX1-20 comprising the first 20 amino acids from the native toxin (IC50 approximately 5 microM), while less effective was the hexapeptide NTX1-6, from the first six amino acid residues of NTX (IC50 = 500 microM). This was the minimum sequence required to block the channel. By testing overlapping sequences from the entire molecule, specially those corresponding to the N-terminal region of NTX, we have been able to determine their different apparent affinities for the KCa channel. Synthetic peptides from the C-terminal region produced no effect on the KCa channel at the concentrations tested (up to 1 mM). These results confirm that in the N-terminal region of the NTX is located part of the sequence that may recognize K+ channels, as we have suggested previously from in vivo experiments. The blockade induced by native NTX was poorly affected by changes in membrane potential; however, the blockage induced by synthetic peptides lacking the C-terminal region was partially released by depolarization.

Amino Acid Sequence↗
PubMed1993

cAMP-dependent phosphorylation modulates voltage gating in an endothelial Cl- channel.

Using the patch clamp technique in the cell-attached, inside-out, and outside-out configurations, we have identified a voltage-gated outwardly rectifying, large conductance (400 pS) Cl- channel in patches from the surface membrane of a cultured monolayer of bovine aortic endothelial cells. The channel is activated in cell-attached patches with 1 microM isoproterenol or 1 mM dibutyryladenosine 3',5'-cyclic monophosphate. In excised inside-out patches the voltage dependence of this channel could be fitted by a Boltzmann distribution with a half-activation voltage (V1/2) at 0 mV. Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphorylation induces a shift of -50 mV in V1/2. Alkaline phosphatases restores the voltage dependence of the channel to control values. The channel is reversibly blocked by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. The half-inhibitory concentration was approximately 70 microM. Our results suggest that beta-adrenergic stimulation (which increases intracellular cAMP levels in this endothelium) may increase Cl- permeability at the cell resting potential by shifting the voltage dependence of this channel.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1993

Depletion and refilling of intracellular Ca2+ stores induce oscillations of Ca2+ current.

An agonist-induced Ca2+ influx pathway in vascular endothelium and other nonexcitable cells is closely aligned with the depletion of microsomal Ca2+ stores. The mechanism by which this occurs is unknown. In these studies 2',5',-di(tert-butyl-1,4-benzohydroquinone, a specific inhibitor of the microsomal Ca(2+)-adenosinetriphosphatase, and patch-clamp recordings were used to evaluate the relationship of inward Ca2+ current to the depletion of intracellular Ca2+ stores. The results demonstrate that depletion and refilling of Ca2+ stores control the amplitude of an electrogenic influx pathway in vascular endothelium. Prominent fluctuations in Ca2+ current occur when there is an imbalance between depletion and refilling of the stores. Furthermore, the studies suggest that the Ca2+ influx pathway is spatially in close association with the intracellular store.

Animals↗
PubMed1993

Cellular basis for the negative inotropic effects of tumor necrosis factor-alpha in the adult mammalian heart.

To define the mechanism(s) responsible for the negative inotropic effects of tumor necrosis factor-alpha (TNF alpha) in the adult heart, we examined the functional effects of TNF alpha in the intact left ventricle and the isolated adult cardiac myocyte. Studies in both the ventricle and the isolated adult cardiac myocyte showed that TNF alpha exerted a concentration- and time-dependent negative inotropic effect that was fully reversible upon removal of this cytokine. Further, treatment with a neutralizing anti-TNF alpha antibody prevented the negative inotropic effects of TNF alpha in isolated myocytes. A cellular basis for the above findings was provided by studies which showed that treatment with TNF alpha resulted in decreased levels of peak intracellular calcium during the systolic contraction sequence; moreover, these findings did not appear to be secondary to alterations in the electrophysiological properties of the cardiac myocyte. Further studies showed that increased levels of nitric oxide, de novo protein synthesis, and metabolites of the arachidonic acid pathway were unlikely to be responsible for the TNF alpha-induced abnormalities in contractile function. Thus, these studies constitute the initial demonstration that the negative inotropic effects of TNF alpha are the direct result of alterations in intracellular calcium homeostasis in the adult cardiac myocyte.

Animals↗
PubMed1992

Anion and cation permeability of a large conductance anion channel in the T84 human colonic cell line.

A large conductance multi-state channel was identified and characterized in single channel recordings from cell-attached and excised patches of the human colonic tumor cell line, T84. The channel activity was dependent on the presence of both permeable cations and anions. In Na(+)-free symmetrical Cl- solutions or Cl(-)-free symmetrical Na+ solutions the channel was inactive. Addition of 5 mM NaCl (NaI or KCl) induced channel activity. The selectivity sequence obtained from the shift in reversal potential was I- (1.9) > Cl- (1) > Na+ (0.5) > K+ (0.3). SO2-(4), SCN- (thiocyanate) and NMDG+ were impermeant. Multiple subconductance states were identified at all voltages explored (+/- 90 mV). The minimum conductance encountered in symmetrical 100 mM NaCl was a 15 pS substrate, the maximum, 210 pS. The channel appeared to be composed of multiples of the 15 pS subunits which were reversibly blocked by the loop diuretic bumetanide (5 microM).

Anions↗
PubMed1992

G-protein-mediated regulation of a Ca(2+)-dependent K+ channel in cultured vascular endothelial cells.

The purpose of the present study was to determine the mechanism by which bradykinin activates the small conductance, inwardly rectifying, Ca(2+)-activated K+ channel (KCa) found in cultured bovine aortic endothelial cells. Channel activity was studied using the patch-clamp technique in whole-cell, cell-attached, inside-out and outside-out configurations. Channel conductance at potentials positive to 0 mV was 10 +/- 2 pS and at potentials negative to 0 mV 30 +/- 3 pS (n = 7) when examined in symmetrical K+ (150 mmol/l) solutions. The channel open probability (P(o)) was only weakly voltage dependent changing approximately 0.2 units over 160 mV. In contrast, raising the intracellular Ca2+ concentration from 100 nmol/l to 10 mumol/l at -60 mV produced a graded increase in channel P(o) from 0.15 to 0.96; the concentration required for half-maximum response (apparent K0.5) was 719 nmol/l. At a constant Ca2+ concentration, application of guanosine triphosphate (GTP) to the cytoplasmic surface of the patch increased channel P(o). This effect was dependent upon the simultaneous presence of both GTP and Mg2+, and was reversed by the subsequent application of the guanosine diphosphate (GDP) analogue, guanosine-5'-O-(2-thiodiphosphate) (GDP beta S). The hydrolysis-resistant GTP analogue, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), induced a long-lasting increase in channel P(o). In the presence of Mg(2+)-GTP, the apparent K0.5 for Ca2+ decreased from a control value of 722 nmol/l to 231 nmol/l. Addition of bradykinin to outside-out patches previously exposed to intracellular Mg(2+)-GTP further enhanced KCa activity, shifting the apparent K0.5 for Ca2+ from 228 nmol/l to 107 nmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Calcium-activated potassium channels from coronary smooth muscle reconstituted in lipid bilayers.

This work is the initial characterization of Ca(2+)-activated K+ (KCa) channels from coronary smooth muscle reconstituted into lipid bilayers. The channels were obtained from a surface membrane preparation of porcine coronary smooth muscle. KCa channels were the predominant K+ channels in this preparation. The conductance histogram (n = 137 channels) revealed two main populations of "maxi" KCa channels with conductances of 245 and 295 pS. Each population could be subdivided in two "isoforms" or "isochannels" with different functional properties (voltage and Ca2+ sensitivities and kinetics). The analysis of "burst" probability of opening showed that at pCa 4 the two isochannels of 245 pS (KCa-1 and KCa-1') had half-activation potentials (V1/2) of -80 and 6 mV, respectively. The isochannels of 295 pS (KCa-2 and KCa-2') had V1/2 of -28 and -66 mV, respectively. KCa-1 had the highest Ca2+ sensitivity; at -60 mV, the concentration of half-activation value for Ca2+ was 1.2 +/- 0.3 microM (n = 5). External tetraethylammonium reduced channel amplitude in a voltage-dependent manner; dissociation constant was 180 +/- 6 and 466 +/- 41 microM at -40 and +80 mV, respectively (n = 5). Charybdotoxin (5-50 nM) produced typical long closings. These effects were similar in all the channels. We conclude that coronary smooth muscle possesses isoforms of maxi KCa channels with Ca2+ and voltage sensors with different properties, which may confer to each channel a specific functional role.

Action Potentials↗
PubMedDate not supplied

[Listeria spp. in food of animal origin].

Different food samples of animal origin were analyzed for Listeria spp. Five L. innocua strains, one L. monocytogenes strain and one L. welshimeri strain were obtained from 208 samples of raw milk. The strains were typified by biochemical and serologic tests. The shortened enrichment method was chosen for isolations; Palcam and Oxford agar also permitted the growth of the seven strains. L. monocytogenes was recovered from milk of an animal with subclinical mastitis. No Listeria strains were isolated from pasteurized milk, chocolate milk or cheese samples. One L. welshimeri strain was detected in ice cream. In the case of meat food samples, the employment of two-step enrichment methods facilitated the detection of Listeria spp. A prevalence of L. ivanovii was observed in 2.5% of these samples.

Animals↗
PubMedDate not supplied

DNA fingerprinting by ERIC-PCR for comparing Listeria spp. strains isolated from different sources in San Luis, Argentina.

In this study, a total of 24 Listeria spp. strains were analyzed. Twenty-two isolates were obtained in San Luis (Argentina) from human, animal, and food samples. Two types of strains, Listeria monocytogenes CLIP 22762 and Listeria innocua CLIP 74915, were included as reference strains. All isolates were biochemically identified and characterized by serotyping, phage typing, and amplification of the flaA gene by polymerase chain reaction (PCR). Repetitive intergenic consensus (ERIC) sequence-based PCR was used to generate DNA fingerprints. On the basis of ERIC-PCR fingerprints, Listeria spp. strains were divided into three major clusters matching origin of isolation. ERIC-PCR fingerprints of human and animal isolates were different from those of food isolates. In addition, groups I and II included ten L. monocytogenes strains, and only one Listeria seeligeri strain. Group III included nine L. innocua strains and four L. monocytogenes strains. Computer evaluation of ERIC-PCR fingerprints allowed discrimination between the tested serotypes 1/2b, 4b, 6a, and 6b within each major cluster. The index of discrimination calculated was 0.94. This study suggests that the ERIC-PCR technique provides an alternative method for the identification of Listeria species and the discrimination of strains within one species.

Animals↗