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Properties of the voltage-dependent calcium channel of mouse Swiss 3T3 fibroblasts.
No full text1. 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
Voltage-dependent calcium current in adherent mouse 3T3 fibroblasts.
No full textWhole-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
Cytosolic calcium and membrane conductance in response to platelet-derived growth factor and bradykinin stimulation in single human fibroblasts.
No full textBradykinin (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
The guanine nucleotide exchange factor Ras-GRF1 directly binds microtubules via DH-PH2 mediated interaction
No full textA voltage-dependent calcium current in mouse Swiss 3T3 fibroblasts.
No full textPatch-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
The neuron specific Ras-exchange factor Ras-GRF1 assembles with polymerized tubulin: microscopy analysis and biochemical studies
No full text
Ras-GRF1, a neuron specific guanine nucleotide exchange factor acting both on Ras and Rac, interacts with microtubules
No full textModulation of extracellular signal-regulated kinases cascade by cronic Delta(99-tetrahydrocannabinol treatment
No full textAcute Delta(9)-tetrahydrocannabinol (THC) injection increased ERK pathway (ERK, pCREB, and c-fos) mostly in the caudate putamen and cerebellum. This effect underwent to homeostatic adaptation after chronic treatment. Moreover, chronic THC exposure induced increases in the ERK cascade (ERK, pCREB, and Fos B) in the prefrontal cortex and hippocampus, suggesting that different neuronal circuits seem to be involved in the early phase and late phase of exposure. The involvement of ERK pathway in cannabinoid chronic exposure was also confirmed in Ras-GRF1 knock out mice, a useful model where cannabinoid-induced ERK activation is lost. In fact, Ras-GRF1 ko mice did not develop tolerance to THC analgesic and hypolocomotor effect. Our data suggest that ERK cascade could play a pivotal role in the induction of synaptic plasticity due to cannabinoid chronic exposure
Serum induces the immediate opening of Ca2+-activated channels in quiescent human fibroblasts.
No full textApplication 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
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