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E Rojas

Publications and source records attributed to E Rojas.

At least 163 records · Page 9Linked to original sources
PubMed1989

Characteristics of two types of calcium channels in rat pituitary gonadotrophs.

The properties of Ca2+ channels in cultured rat pituitary gonadotrophs were analyzed by the patch-clamp technique. The inward Ca2+ currents, recorded in the presence of 5.2 mM Ca2+ or Ba2+, included a fast, transient component with activation-inactivation kinetics and a delayed component with slower activation. The midpoint of the activation curve lay at -30 mV for the transient component and at -12 mV for the delayed component. At the midpoint, changes in potential of 9.5 and 13 mV induced an e-fold change in the activation of the transient and delayed components, respectively. The rate of inactivation of the first component was strongly voltage dependent. At -43 mV, a 7.4-mV change in potential induced an e-fold change in the fraction of Ca2+ channels available to conduct Ca2+ current. During long-lasting (100-200 ms) low-frequency depolarizing voltage-clamp pulses, the size of the delayed component of the Ca2+ current remained constant. The differential effects of membrane potential on inactivation and the different time constants for activation of the two components of the Ca2+ conductance indicate the presence of two types of Ca2+ channels in the membrane of the gonadotroph: the rapidly inactivating current appears to be attributable to a T-type channel, and the noninactivating current corresponds to the L-type channel described in many other cell types.

Animals↗
PubMed1988

The ATP-sensitive potassium channel in pancreatic B-cells is inhibited in physiological bicarbonate buffer.

The effects of bicarbonate buffer (HCO3-/CO2) on the activity of the two K+ channels proposed by some to control the pancreatic B-cell membrane response to glucose were studied. Single K+-channel records from membrane patches of cultured B-cells dissociated from adult rat islets exposed to a glucose- and bicarbonate-free medium (Na-Hepes in place of bicarbonate) exhibit the activity of both the ATP-sensitive as well as the [Ca2+]i-activated K+ channels. However, in the presence of bicarbonate-buffered Krebs solution, the activity of the ATP-sensitive K+ channel is inhibited leaving the activity of the K+ channel activated by intracellular [Ca2+]i unaffected. In the absence of bicarbonate (Hepes/NaOH in place of bicarbonate), lowering the external pH from 7.4 to 7.0 also has differential effects on the two K+ channels. While the K+ channel sensitive to ATP is inhibited, the K+ channel activated by a rise in [Ca2+]i is not affected. To determine whether the response of the B-cell in culture to bicarbonate is also present when the B-cell is functioning within the islet syncytium, the effects of bicarbonate removal on membrane potential of B-cells from intact mouse islets were compared. These studies showed that glucose-evoked electrical activity is also blocked in bicarbonate-free Krebs solution. Furthermore, in the absence of bicarbonate and presence of glucose (11 mM), electrical activity was recovered by lowering the pHo from 7.4 to 7.0. The ATP-sensitive K+-channel activity is greatly reduced by physiologically buffered solutions in pancreatic B-cells in culture. The most likely explanation for the bicarbonate effects is that they are mediated by cytosolic pH changes. Removal of bicarbonate (keeping the external pH at 7.4 with Hepes/NaOH as buffer) would increase the pHi. Since the activity of the [Ca2+]i-dependent K+ channels is not affected by the removal of the bicarbonate buffer, our patch-clamp data in cultured B-cells indicate an involvement of [Ca2+]i-activated K+ channels in the control of the membrane potential. For the B-cell in the islet, we propose that the burst pattern of electrical activity (Ca2+ entry) is controlled, at least in part, by the [Ca2+]i-activated K+ channel.

Adenosine Triphosphate↗
PubMed1988

Differential effects of maitotoxin on ATP secretion and on phosphoinositide breakdown in rat pheochromocytoma cells.

Maitotoxin (MTX) induced exocytotic secretion of ATP from PC12 rat pheochromocytoma cells. The threshold for stimulation of secretion was at concentrations of about 2 ng/ml of MTX. Maximal release occurred at 40 ng/ml. MTX-induced ATP release required the presence of calcium in the extracellular medium and could be inhibited by nifedipine, a specific blocker of voltage-dependent calcium channels. In addition to the effects on ATP secretion from PC12 cells, MTX stimulated the breakdown of phosphoinositides, as measured by the accumulation of [3H]inositol phosphates. Maximal stimulation of phosphoinositide breakdown was reached at only 0.5-1.0 ng/ml MTX. MTX at concentrations required to evoke ATP release (greater than 2 ng/ml) had lesser or no effect on phosphoinositide breakdown. Although stimulation of phosphoinositide breakdown by MTX was dependent on extracellular calcium, it was insensitive to the calcium channel blockers nifedipine, D-600 and cobalt ions. The different concentration range required to elicit these responses and the varying sensitivity to calcium channel blockers indicate that MTX-evoked secretion and MTX-stimulated phosphoinositide breakdown are independent phenomena in PC12 cells.

Adenosine Triphosphate↗
PubMed1988

Use of selective toxins to separate surface and tubular sodium currents in frog skeletal muscle fibers.

The interaction between toxin gamma from the venom of the scorpion Tityus serrulatus and sodium channels in skeletal muscle membranes from the frog Caudiverbera caudiverbera was studied. Sodium current from cut sartorius muscle fibers is a complex signal in which early and late components are difficult to separate. External application of Tityus gamma toxin initially blocked the early component in a voltage-dependent manner. Longer exposure to the toxin induced a complete blockade of the two components of the inward current. Application of tetrodotoxin to fibers pretreated with Tityus toxin at submaximal concentrations allowed the observation of the two distinct components of the inward current. Binding of 125I-labelled toxin to highly purified membrane fractions from the same muscle was used to establish the presence of high affinity receptors both in the transverse-tubular and in the surface membrane.

Animals↗
PubMed1988

An alternative in vitro method for testing the potency of the polyvalent antivenom produced in Costa Rica.

The ability of several batches of polyvalent antivenom to neutralize indirect hemolytic activity of Bothrops asper venom was studied using a sensitive plate test. All samples of antivenom tested effectively neutralized this activity. A highly significant correlation was observed between neutralization of indirect hemolysis and neutralization of lethal activity. This simple and sensitive in vitro test could be used to monitor antibody levels in horses immunized to produce polyvalent antivenom.

Animals↗
PubMed1988

Ca2+-activated synexin forms highly selective, voltage-gated Ca2+ channels in phosphatidylserine bilayer membranes.

Synexin, a cytosolic protein that mediates Ca2+-dependent membrane fusion, was incorporated into acidic phospholipid bilayers, formed at the tip of a patch pipet. The pipet was filled with a high-Ca2+ solution (50 mM) and immersed in a chamber containing a low-Ca2+ solution (1 mM). Brief exposures of the bilayer to synexin increased the capacitance of the bilayer by a factor of 10 and decreased the membrane resistance by a factor of 20. Reduction of Ca2+ in the chamber to 1 microM caused an abrupt increase in the current required to hold the pipet potential at 0 mV. Under certain conditions channel events could be detected, often occurring in bursts. Consistently, open-time histograms were found to be voltage-dependent and to exhibit one time constant in the time range examined here. The slope conductance for the synexin channel was estimated as 10.2 +/- 2.1 pS for the large Ca2+ gradient with low chamber Ca2+. However, for symmetrical, low-Cl- solutions containing 25 mM Ca2+ the conductance was 26.5 +/- 5.2 pS. Ion-replacement studies showed the synexin channel to much prefer Ca2+ over Ba2+ or Mg2+. Cd2+, a potent blocker of other voltage-gated Ca2+ channels at 100 microM, blocked synexin channels only at very high concentrations (greater than or equal to 10 mM). Similarly, nifedipine, an inhibitor of the nonactivating Ca2+ channel, was effective only at extremely high concentrations (greater than 300 microM). The high selectivity for Ca2+ and the lack of response of the channel to various drugs known to block Ca2+ channels thus distinguish the synexin channel from other types of Ca2+ channels hitherto reported.

Animals↗
PubMed1988

Electrical activity in chromaffin cells of intact mouse adrenal gland.

Membrane potentials of medullary chromaffin cells of the adrenal gland of the mouse were measured in situ. Resting potential (-54.3 +/- 8.8 mV) depended on extracellular [K+] as predicted by the constant-field equation with a permeability ratio, PNa/PK, of 0.09. Current-voltage (I-V) relationships showed that the current is rectified across the chromaffin cell membrane. A rectification ratio of 0.4 was calculated from the slopes of the I-V curves for positive (41 +/- 26 M omega) and negative (103 +/- M omega) currents. Because input resistance for a resting chromaffin cell in isolation is approximately 5 G omega, the chromaffin cells in situ behave as if they were electrically coupled. Most cells responded to depolarizing current pulses with repetitive action potentials, but only 50% of them showed spontaneous electrical activity. Spontaneous activity was often seen in the presence of tetrodotoxin (3 microM). Although the application of the K+-channel blockers tetraethylammonium and Ba2+ greatly increased the amplitude of the action potentials, only Ba2+ induced continuous electrical activity. Application of acetylcholine (ACh) always depolarized the cell membrane. This effect was blocked by atropine but not by D-tubocurarine, suggesting that ACh stimulation of chromaffin cells in the mouse involves activation of muscarinic receptors.

Acetylcholine↗
PubMed1988

A molecular basis for synexin-driven, calcium-dependent membrane fusion.

Membranes of secretory vesicles fuse with each other and with plasma membranes during exocytosis in many different cell types. The probable role of calcium in the process is now widely accepted, and it is possible that at least one cytosolic mediator of calcium action is synexin. Synexin is a 47,000 Mr calcium-binding protein, initially discovered in the bovine adrenal medulla, which binds to granule membranes and to inner aspects of chromaffin cell plasma membranes. Synexin causes chromaffin granules to aggregate, and such aggregates can be caused to fuse in the additional presence of arachidonic acid. Synexin also mediates the direct fusion of liposomes and chromaffin granule ghosts. To understand better the mechanisms of membrane fusion promoted by synexin we have attempted to define the primary sequence of the protein. Our initial efforts were directed towards purification of bovine synexin in sufficient amounts to allow us to sequence tryptic peptides. However, as the project progressed we also directed our attention to human synexin, preparing peptides from this protein as well. From analysis of bovine peptides we learned that the synexin molecule might be closely related to a class of proteins including lipocortin I, calpactin (p36), endonexin II, protein II and calelectrin 67K. Complete analysis of a human synexin cDNA clone revealed strong homology with bovine synexin. The analysis also showed that synexin contained a unique, long, highly hydrophobic N-terminal leader sequence followed by a characteristic four-fold repeat homologous with those found in other members of the synexin gene family. The highly hydrophobic character of synexin seems consistent with information previously obtained that synexin is able to insert directly into the interior of bilayers prepared not only from purified phosphatidylserine but also from biological membranes. The evidence for such insertions is a dramatic increase in the capacitance of the membrane, formed at the tip of a patch pipette, when calcium-activated synexin is applied to the bilayer. Additional evidence is the fact that synexin also forms calcium-selective channels when the protein is applied to the cytosolic aspect of the plasmalemma when that side is also exposed to calcium at sub-millimolar concentrations. Thus, the synexin molecule not only enters the membrane, but also spans it. From these and other data we have developed the concept that the fusion process may involve synexin forming a 'hydrophobic bridge' between two fusing membranes. Lipid movement across this bridge may then be the material basis for final fusion.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Production of monovalent anti-Bothrops asper antivenom: development of immune response in horses and neutralizing ability.

A monovalent antivenom was produced by immunizing two horses with venom of the pit viper Bothrops asper (Ophidia: Viperidae). Although development of the immune response against four toxic and enzymatic activities of the venom was similar in both horses during the first two thirds of the immunization schedule, antibody response in one of the horses reached much higher levels in the last part of the immunization. Immunoelectrophoretic analysis indicates that there were precipitating antibodies in the sera of these horses during all the stages of immunization. However, immunoprecipitation did not correlate with the ability of sera to neutralize toxic activities of B. asper venom. Monovalent antivenom was more effective than the commercially available polyvalent antivenom in the neutralization of Bothrops asper venom. On the other hand, despite the fact that it neutralizes lethal and hemorrhagic activities of the venoms of Lachesia muta and Crotalus durissus durissus, it was less effective than polyvalent antivenom in these neutralizations. Moreover, it does not neutralize defibrinating activity induced by these two venoms, whereas it neutralizes this effect in the case of B. asper venom. It is proposed that monovalent antivenom may be highly effective in the case of envenomations induced by Bothrops asper venom; its use in treating accidents by L. muta and C. durissus would be indicated only if polyvalent antivenom is not available. Results also demonstrate that it is important to monitor antibody response individually in horses being immunized for antivenom production, due to the conspicuous variability in the response of different animals.

Animals↗
PubMed1987

Evidence for modulation of cell-to-cell electrical coupling by cAMP in mouse islets of Langerhans.

The effects of forskolin on electrical coupling among pancreatic beta-cells were studied. Two microelectrodes were used to measure membrane potentials simultaneously in pairs of islet beta-cells. Intracellular injection of a current pulse (delta I) elicited a membrane response delta V1 in the injected cell and also a response delta V2 in a nearby beta-cell confirming the existence of cell-to-cell electrical coupling among islet beta-cells. In the presence of glucose (7 mM), application of forskolin evoked a transient depolarization of the membrane and electrical activity suggesting that the drug induced a partial inhibition of the beta-cell membrane K+ conductance. Concomitant with this depolarization of the membrane there was a marked decrease in beta-cell input resistance (delta V2/delta I) suggesting that exposure to forskolin enhanced intercellular coupling. Direct measurements of the coupling ratio delta V2/delta V1 provided further support to the idea that forskolin enhances electrical coupling among islet cells. Indeed, application of forskolin reversibly increased the coupling ratio. These results suggest that cAMP might be involved in the modulation of electrical coupling among islet beta-cells.

Adenylyl Cyclases↗
PubMed1987

Membrane capacity measurements suggest a calcium-dependent insertion of synexin into phosphatidylserine bilayers.

The mechanism by which synexin mediates calcium-dependent aggregation of medullary cell chromaffin granules and fusion of granule ghosts involves specific interactions with the lipid component of the membrane. To study the details of these interactions we measured synexin-induced changes in capacitance of phosphatidylserine bilayers formed at the tip of a patch pipet using the double-dip method. Provided calcium was present in the solution filling the pipet (10-50 mM) stable phosphatidylserine bilayers were easily formed. Addition of synexin (0.1 microgram/ml) to an external medium lacking added calcium induced no measurable changes in either bilayer resistance (10-30 G omega) or displacement current across the membrane. However, addition of calcium (0.1-2.5 mM) in the presence of synexin in the external solution caused a marked increase in the size and time constant of decay of the displacement current. From the steady-state value of the current we calculated a 5-fold decrease in resistance and from the charge displaced during the voltage-clamp pulses we calculated a 10-fold increase in membrane capacitance (from 20 to 200 fF). The size of the synexin-specific charge displacement in one direction during a pulse was always equal to the charge returning to the original configuration after the pulse. The synexin-specific transfer of charge reached saturation when the pipet potential was taken to a sufficient positive or negative value. These properties of the extra charge movement support our view that in the presence of calcium the cytosolic protein synexin penetrates into the bilayer. It is possible that these properties may be related to the mechanism by which synexin promotes membrane fusion in natural membranes.

Annexin A7↗
PubMed1987

Inositol 1,4,5-trisphosphate-induced Ca2+ release from the sarcoplasmic reticulum and contraction in crustacean muscle.

Intracellular applications of a fixed amount (0.2 to 8 nmol) of inositol 1,4,5-trisphosphate (InsP3) over a brief period (2 s) into barnacle muscle fibers induced vigorous contractures. Peak tension attained during the first application depended on [InsP3]: the maximum tension evoked by the injection of 8 nmol was 1.6 kg/cm2. Peak tension during a second application of a high dose of InsP3 (greater than 10 microM) was always smaller than that during the first application. Extracellular Ca2+ could be omitted with no measurable effects on either the amplitude or time course of the contractures evoked by InsP3. Aequorin was used to measure InsP3-evoked Ca2+ release from intracellular stores in minced muscle fibers from lobster and in skinned muscle fibers from barnacle. Provided the sarcoplasmic reticulum was preloaded with Ca2+, application of InsP3 induced a transient Ca2+ release that was [InsP3] dependent. During each transient, [Ca2+] rose rapidly to a peak value (t1/2 less than 5 s) and then slowly returned (t1/2 less than 100 s) to a basal level. Maximum Ca2+ release was obtained at [InsP3] less than 100 microM and amounted to 4 nmol Ca2+/g of muscle, enough to increase [Ca2+]i from 0.1 to 8 microM had the Ca2+ release occurred in the intact fiber. Successive applications of a fixed amount of InsP3 elicited successive transient increases in Ca2+. The effects of [Ca2+] on the incorporation of [3H]inositol into the pools of phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate pools were measured.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Modulation of K+ conductance by intracellular pH in pancreatic beta-cells.

Measurements of the effects of NH3/NH4+ on glucose-induced electrical activity in beta-cells from microdissected mouse islets of Langerhans and on intracellular pH in single collagenase-isolated islets pre-loaded with a fluorescent pH probe were performed and are reported here. Application of NH3/NH4+ (15 mM) in the presence of glucose (11 mM) promptly hyperpolarized the beta-cell membrane, reduced input resistance by 60% and blocked electrical activity. These changes were paralleled by an increase in islet fluorescence indicative of a cytosolic pH increase. Removal of NH4Cl initially stimulated electrical activity, which returned to resting level with a time constant of 51 s. Concomitant with the removal of NH4Cl there was a drop in pHi followed by a slow return to resting level with a time constant of 83 s. The results suggest that the [Ca2+]-dependent K+ channel in the beta-cell membrane is activated by a rise in cytosolic pH.

Ammonium Chloride↗
PubMed1986

Muscarinic receptor enhancement of nicotine-induced catecholamine secretion may be mediated by phosphoinositide metabolism in bovine adrenal chromaffin cells.

Bovine adrenal chromaffin cells possess both nicotinic and muscarinic cholinergic receptors, but only nicotinic receptors have heretofore appeared to mediate Ca2+-dependent exocytosis. We have now found that muscarinic receptor stimulation in bovine adrenal chromaffin cells leads to enhanced inositol phospholipid metabolism as evidenced by the rapid (less than 1 min) formation of inositol trisphosphate (IP3) and inositol bisphosphate (IP2). Muscarinic receptor-mediated accumulation of IP3 and IP2 continues beyond 1 min in the presence of LiCl and is accompanied by large increases in inositol monophosphate. Muscarinic receptor stimulation was also found to enhance nicotine-induced catecholamine secretion by 1.7-fold if muscarine was added 30 s before nicotine addition. Moreover, since the muscarinic antagonist atropine reduces acetylcholine-induced secretion, we conclude that muscarinic receptor stimulation somehow primes these cells for nicotinic receptor-mediated secretion, perhaps by causing small nonstimulatory increases in cytosolic free Ca2+ mediated by IP3. Furthermore, we show that small depolarizations of these cells with 10 mM K+, which themselves do not affect basal secretion, also enhance nicotine-induced secretion. Thus, small increases in cytosolic free Ca2+ produced either by physiologic muscarinic receptor stimulation or by small experimental depolarizations with K+ may prime the chromaffin cells for nicotinic receptor-mediated secretion.

Adrenal Medulla↗
PubMed1986

Potassium channel selectivity in mouse pancreatic B cells.

High-resistance microelectrodes were used to measure membrane potential changes in response to increased extracellular K+ concentration ([K+]o; or a test cation X+ such as Li+, Rb+, Cs+, NH+4) in B cells from mouse islets of Langerhans. In the absence of glucose, a sudden increase in [K+]o (or [X+]o), keeping the sum [Na+]o + [K+]o constant (or [Na+]o + [K+]o + [X+]o), induced a rapid depolarization of the membrane. The membrane potential changes were essentially unchanged in the presence of 20 mM tetraethylammonium (TEA). The Goldman-Hodgkin-Katz equation was fitted to the experimental relationship between membrane potential and [K+]o (or [X+]o), and permeability (P) ratios were estimated. In the absence of TEA, P Na/PK was estimated to be approximately 0.046. In the presence of TEA the following ratios were estimated: P Rb/PK = 0.74, P Cs/PK = 0.62, and P NH4/PK = 0.36. From these ratios the following sequence of permeabilities was obtained, PK greater than P Rb greater than P Cs greater than P NH4 greater than P Na. It is proposed that this sequence reflects the selectivity of the intracellular [Ca2+]-activated K+ channel of the pancreatic B cell.

Ammonia↗