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A Peres

Publications and source records attributed to A Peres.

At least 73 records · Page 4Linked to original sources
PubMed1990

Mitogen-induced oscillations of membrane potential and Ca2+ in human fibroblasts.

Using the whole-cell technique, we have measured recurring hyperpolarizations induced by fetal calf serum and bradykinin in human fibroblasts. By coupling fura-2 microfluorimetry to electrophysiology, we have also measured directly cytosolic Ca2+ and found that Ca2+ oscillations occur in synchrony with membrane currents. Mitogen stimulation of cells in which intracellular K+ had been replaced with Cs+ resulted in the abolishment of the outward current. We conclude then that the mitogen-induced recurring hyperpolarizations in human fibroblasts are due to the opening of Ca2(+)-activated K+ channels.

Benzofurans↗
PubMed1990

Cytosolic calcium and membrane conductance in response to platelet-derived growth factor and bradykinin stimulation in single human fibroblasts.

Bradykinin (BK) and platelet-derived growth factor (PDGF) act as mitogens and stimulate phosphatidylinositol (PI) turnover in human fibroblasts. By coupling whole-cell electrophysiological measurements with cytosolic Ca2+ determinations using fura-2 microfluorimetry, we have studied the changes in cytosolic calcium and in membrane conductance in single cells following stimulation with BK or PDGF. Both agonists produce variable patterns of response which include: single transient, sustained pulsations, damped oscillations, no response. In all cases, there is a very good temporal correlation between increases in intracellular Ca2+ and membrane current. The cytosolic calcium elevation appears to be insensitive to membrane potential changes, indicating that Ca2+ is released from an intracellular source. The Ca2(+)-activated current is not blocked by 1 microM apamin or by 0.5 mM (+)-tubocurarine; it is instead strongly reduced by 5 mM tetraethylammonium (TEA). We can conclude that BK and PDGF induce very similar early responses in human fibroblasts, and that the variable pattern of response does not depend on the particular mitogen used. The membrane currents are due to a kind of Ca2(+)-activated K+ channels which, according to their voltage-dependence and specific blockers, belong to the "maxi K+" class.

Bradykinin↗
PubMed1989

Voltage-dependent calcium current in adherent mouse 3T3 fibroblasts.

Whole-cell recording was performed on adherent mouse Swiss 3T3 fibroblasts. Depolarizations from a holding potential of -100 mV gave rise to a transient inward current. The voltage dependence, kinetic properties, and ionic selectivity of this current are identical to those described in the same cells kept in suspension after detachment from the culture dish [A. Pandiella, A. Malgaroli, J. Meldolesi, and L.M. Vicentini (1987) Exp. Cell. Res. 170, 175-185; W.H. Moolenaar, L.G.J. Tertoolen, and S.W. de Laat (1984) J. Biol. Chem. 259, 8066-8069].

Action Potentials↗
PubMed1989

Membrane conductance oscillations induced by serum in quiescent human skin fibroblasts.

1. Application of fetal calf serum to quiescent human fibroblasts, kept under whole-cell voltage clamp at positive potentials, induced a series of transient rises in membrane conductance. 2. The first transient increase in conductance developed with very short time lag (2-10 s) after serum addition, while the period between successive transients was 30-90 s, being remarkably constant in each particular cell. 3. Raising the Ca2(+)-buffering capacity of the intracellular solution with 1 mM-EGTA suppressed the appearance of the sustained oscillations. 4. The conductance increase was strongly voltage dependent: voltage ramps applied before, during and after the transients revealed the activation of an outwardly rectifying conductance with variable reversal potentials (between +14 and -55 mV). 5. No significant shifts of the reversal potential were observed when the extracellular K+ concentration was increased to 126 mM. Substitution of K+ with Cs+ as intracellular cation eliminated the outward current in response to serum. 6. External application of the Ca2+ ionophore A23187 elicited currents which were very similar in voltage dependence and time course to those triggered by serum. 7. The serum-induced response persisted unaffected by the absence of external Ca2+. The response was also seen in the presence of 1 mM-Cd2+ in the external solution. 8. Serum addition caused a rapid morphological rearrangement of the cells. 9. It is concluded that serum triggers a mobilization of Ca2+ from intracellular stores which in turn activates cationic channels.

Blood Physiological Phenomena↗
PubMed1989

The effects of polyinosinic:polycytidylic acid on the graft-versus-host reaction. III: Increased severity of the reaction with delayed pI:C treatment.

We have been investigating the effects of polyinosinic:polycytidylic acid (pI:C), an interferon inducer, on the graft-versus-host reaction. We have previously shown that pI:C treatment of C57BL/6xAF1 (B6AF1) recipient mice immediately before injection of C57BL/6 (B6) parental lymphocytes inhibited the immuno-suppression and pathological changes normally caused by the GVH reaction, by a mechanism apparently identical to that seen in F1 hybrid resistance (HR) to hematopoietic grafts. We now demonstrate that delaying pI:C treatment by as little as 48 hr produces the opposite effect. Treatment of recipient B6AF1 mice at different days after transfer of parental lymphocytes induced a marked increase in the severity of the GVH reaction, as measured by a decreased plaque-forming cell response to sheep erythrocytes; decreased proliferative response to the T and B cell mitogens PHA, Con A, and LPS; increased pathological changes in both lymphoid and nonlymphoid tissues; and increased GVH-associated mortality. This effect is unrelated to HR, as pI:C was able to augment the severity of the GVH reaction when A strain cells were injected into AxCBAF1 recipients, which do not manifest HR. Early pI:C treatment (1 and 2 days after parental cell transfer) increased the severity of the GVH reaction much more than later pI:C treatment (7 and 8 days after parental cell transfer). This observation, along with the demonstration of altered pathology in GVH mice treated with pI:C, suggests that the effect of pI:C is not mediated through a direct suppressive effect of IF on the cells responding in either the PFC or mitogen assays, but rather by the ability of IF to activate or suppress mechanisms involved in the development of GVH-induced alterations.

Animals↗
PubMed1988

Serum induces the immediate opening of Ca2+-activated channels in quiescent human fibroblasts.

Application of fetal calf serum to quiescent human fibroblasts produces an immediate (3-20 s delay) increase in membrane conductance which lasts about 20-30 s. This conductance is strongly outwardly-rectifying and has a reversal potential between -45 and -10 mV. The conductance increase may also be induced by application of the Ca2+ ionophore A23187 while it does not occur when intracellular K+ is replaced by Cs+. It is concluded that this early effect of serum is due to the opening of Ca2+-activated channels. This permeability change will alter the membrane potential and thus possibly interact with other voltage-sensitive processes induced by serum growth factors.

Blood↗
PubMed1988

A voltage-dependent calcium current in mouse Swiss 3T3 fibroblasts.

Patch-clamp experiments in the whole-cell mode have been performed in Swiss 3T3 mouse fibroblasts. Depolarizations from negative holding potential (Vh less than -60 mV) gave rise to a rapidly activating, fully inactivating, inward current of few tenths of nA in physiological saline at 35 degrees C. The current persisted when external Na+ was replaced by impermeant TMA+ and disappeared in 0 Ca2+, 1 mM EGTA. The current was reversible blocked by Co2+ and it was slightly reduced when external Ca2+ was substituted by Ba2+. Finally its reversal potential changed with Nernstian slope with increasing external Ca2+ concentrations. It is concluded that these cells possess a voltage-dependent Ca2+ channel.

Animals↗
PubMed1988

Properties of the voltage-dependent calcium channel of mouse Swiss 3T3 fibroblasts.

1. Suspended Swiss 3T3 fibroblasts were voltage clamped using the whole-cell technique. 2. Passage from the cell-attached to the whole-cell mode was accompanied by only a minor decrease in input resistance. Direct measurement of resting potential gave values between O and -15 mV. 3. In order to account for the effects of leak on the membrane potential measurements, I-V curves were obtained immediately before and after patch rupture by applying voltage ramps. After subtraction of the cell-attached current from the whole-cell current, the true membrane potential was estimated as the zero-current potential in the I-V curve. An average value of -8.2 +/- 0.9 mV in 8 mM-Ca2+ was obtained in this way. 4. In 2 mM-Ca2+, step depolarizations 100 ms long from holding potentials (Vh) more negative than -60 mV caused a transient inward current to appear. From Vh greater than -60 mV only a linear leakage component was apparent. 5. In 2 mM-Ca2+ depolarizations to potentials greater than +40 mV (from Vh = -100 mV) generated transient, outwardly directed currents. 6. Increasing extracellular Ca2+ up to 32 mM shifted the peak current vs. voltage curve and the reversal potential (Erev) towards more positive potentials, and caused an increase of the peak current. 7. The steady-state inactivation curve was the same for both inward and outward currents, indicating that they flow through the same channels. The currents are completely inactivated at V = -60 mV. 8. Recovery of the fully inactivated current upon hyperpolarization had an exponential time course with tau = 0.22 s at V = -80 mV and tau = 0.18 s at V = -100 mV. 9. In the absence of Ca2+ (but with Mg2+ present) the inward current disappeared but a large, inactivating outward current appeared when V greater than 0 mV. The current was strongly reduced by Cd2+ (1 mM) or Co2+ (10 mM). 10. Complete removal of divalent cations from the external solution caused the channel to become highly permeable to monovalent cations. 11. Nitrendipine (10 microM) and verapamil (5 microM) were unable to block the current. 12. On the whole the present results indicate that voltage-dependent Ca2+ channels are present in these cells. Their sensitivity to divalent cations, to organic blockers and to potential is similar to that of the low-voltage-activated, or 'T' type, Ca2+ channels described in other cells.

Action Potentials↗
PubMed1987

Single- and multi-channel currents recorded with patch electrodes in mouse eggs.

Zona-free mouse eggs are amenable to patch recording as well as to whole-cell recording (Bland et al., 1984; Peres, 1986a). Although many patches are silent, others show spontaneous channel opening; the single channel current is 1.5 pA and inwardly directed at resting potential (RP). Current amplitudes distribute normally as a single population at RP, however at RP-50 mV the amplitude histogram indicates two channel populations. Open time distribution is exponential with a mean open time between 4 and 7 msec at RP. In three cases out of thirty-eight, in which depolarizations were given from a holding potential of RP-50 mV, microscopic currents very similar in kinetics and voltage-dependence to the whole-cell Ca2+ current were observed. No single-channel currents could be resolved in these traces. The results reported here indicate that the voltage-dependent Ca2+ current of the mouse egg goes through low-conductance channels located in high density spots on the egg surface.

Animals↗
PubMed1987

The calcium current of mouse egg measured in physiological calcium and temperature conditions.

1. Voltage-clamp experiments have been performed on ovulated mouse eggs using the whole-cell recording technique. 2. Whole-cell recording offers improved signal-to-noise ratio and excellent stability over time. This allowed the study of the Ca2+ current of these eggs under physiological conditions (i.e. 1.7 mM-external Ca2+ and 37 degrees C). 3. In these conditions a negative shift of the reversal potential of the current (about -25 mV) and also of the activation and inactivation parameters (about -10 mV) compared with those recorded in 20 mM-external Ca2+ is found. 4. No significant diminution of the inward current was detected when external Na+ was substituted with impermeant cations, indicating no relevant participation of Na+ to the current in physiological conditions. 5. In a medium free of divalent cations a large inward current appeared, together with a large decrease in membrane resistance. 6. In Ca2+-free medium containing 1.2 mM-Mg2+ the inward current was largely suppressed, while an outward transient current appeared for depolarizations greater than +10 mV. 7. The 'outward surge current' previously described in this preparation appears to possess the same inactivation time constant and the same steady-state inactivation curve as the inward Ca2+ current. This suggests that the two currents flow through the same channels. 8. The time constant of inactivation was the same for both inward and outward currents and was independent of the current amplitude. These observations exclude a Ca2+-induced type of inactivation. 9. The channel which physiologically carries the Ca2+ current in mouse eggs belongs then to the class of Ca2+ channels that owe their selectivity to high-affinity Ca2+ binding sites.

Action Potentials↗
PubMed1986

The effects of polyinosinic:polycytidylic acid (pI:C) on the graft-vs-host (GVH) reaction. II. Increased NK-mediated rejection on C57BL/6 lymphocytes by (C57BL/6 X A)F1 mice.

We previously demonstrated that treatment of (C57BL/6 X A)F1 (F1) recipient mice with polyinosinic:polycytidylic acid (pI:C) before injection with 30 X 10(6) C57BL/6 (B6) lymphocytes prevents both the immunosuppression and pathologic lesions typical of graft-vs-host (GVH) reactions. We now report the further characterization of this phenomenon. Donor spleen and lymph node cells were labeled with fluorescein in vitro and injected into pI:C-treated or untreated mice. Two days later, recipient splenocytes were analyzed for the presence of fluorescein-labeled donor cells by flow microfluorometry. Treatment of F1 mice with pI:C resulted in a sharp reduction in the recovery of labeled B6 but not A strain parental cells. Treatment with pI:C had no effect when syngeneic recipients were used, or when F1 cells were injected into A, B6, or F1 recipients. These results suggest that pI:C treatment induces rejection of B6 but not A or F1 lymphocytes by F1 hybrid mice at least as early as 2 days after donor cell transfer. As F1 cells are not rejected by either parent, rejection does not seem to be directed against classical alloantigens. These observations are compatible with the previously described model of hybrid resistance (HR) against bone marrow grafts. The rapidity of rejection strongly suggested that natural cytotoxic mechanisms were involved, thus, natural killer (NK) cell and macrophage (M phi) cytotoxic activities were tested throughout the time when the parental cell graft was being rejected. Over this period, pI:C treatment increased cytotoxic activity against the NK-sensitive target cell line YAC-1 but had no effect on spontaneous M phi tumoricidal activity against the L5178Y and MDAY-D2 cell lines. The results suggest that NK cells, but not M phi, may be involved in the elimination of B6 parental cells by the pI:C-treated F1 mice. NK cells have been demonstrated to be radioresistant; thus, as a test of our hypothesis, we examined the effects of irradiation on the capacity of pI:C treated F1 mice to reject B6 lymphocytes. The results show that this capacity was not blocked by 750 cGy, a dose of radiation that abrogates most T and B cell functions. Furthermore, rejection of parental cells could be prevented by treatment of recipient F1 mice with antibodies to asialo GM1, a treatment that suppresses NK activity. These data demonstrate that pI:C-mediated protection from GVH-induced changes is due to increased rejection of grafted B6 parental cells by F1 NK cells, a phenomenon very similar, if not identical, to HR to bone marrow grafts.

Animals↗
PubMed1986

Computer reconstruction of the spread of excitation in nerve terminals with inhomogeneous channel distribution.

A direct numerical integration method, as modified by Du Fort and Frankel (1953), has been used to solve the partial differential equation system which describes the spread of action potential in a mammalian nerve terminal. Branching of the terminal as well as inhomogeneous distributions of Na+ and K+ voltage-dependent channels (Brigant and Mallart 1982) have been incorporated in the model. Using the channel densities and the kinetic parameters measured in the node of Ranvier, the depolarization in the terminal branches has an amplitude of only 60% of the action potential in the node. Furthermore, the time courses of the calculated membrane currents differ considerably from the ones measured by Brigant and Mallart (1982) and by Konishi and Sears (1984). Increasing the Na+ and K+ channel densities may considerably increase the terminal depolarization and also reproduce qualitatively the current wave-forms observed experimentally. The model can also reproduce some of the effects of pharmacological channel blocks. The simulation allows a new interpretation of the different components of membrane current along the terminal.

Action Potentials↗