Biomedical subjects

G Cota

Publications and source records attributed to G Cota.

25 records · Page 2Linked to original sources

PubMed1986

Calcium channel currents in pars intermedia cells of the rat pituitary gland. Kinetic properties and washout during intracellular dialysis.

Ca channel currents in primary cultured pars intermedia cells were studied using whole-cell recording with patch pipettes. Experiments were carried out at 18-21 degrees C in cells internally dialyzed with K-free, EGTA-containing solutions and in the presence of 10 mM Ca or 10 mM Ba in the external solution. Ca and Ba currents depended on the activity of two main populations of channels, SD and FD. With Ca as the charge carrier, these two populations differed in their closing time constants at -80 mV (SD, 1.8 ms; FD, 110 microseconds), apparent activation levels (SD, -40 mV; FD, -5 mV), half-maximal activation levels (SD, +5 to +10 mV; FD, +20 to +25 mV), half-times of activation at +20 mV (SD, 2.5-3.5 ms; FD, 1.0-1.3 ms), and time courses of inactivation (SD, fast; FD, slow). Functional FD channels were almost completely lost within 20-25 min of breaking into a cell, whereas SD channels retained most of their functional activity. In addition, the conductance-voltage curve for FD channels shifted approximately 15 mV toward more negative membrane potentials within 11-14 min under whole-cell recording. At that time, 60-70% of the FD channel maximum conductance was lost. However, the conductance-voltage curve for SD channels shifted less than 5 mV within 25 min. The addition of 3 mM MgATP and 40 microM GTP to the internal solution slowed down the loss of FD channels and prevented the shift in their activation curve. It was also found that the amplitude of the current carried by FD channels tends to increase as a function of the age of the culture, with no obvious changes in the kinetic properties of the channels or in SD channel activity.

Adenosine Triphosphate↗

PubMed1986

A fast-activated inward calcium current in twitch muscle fibres of the frog (Rana montezume).

Voltage-clamp experiments were performed at 18 degrees C in intact twitch muscle fibres of the frog using the three micro-electrode technique. Membrane currents were recorded in the presence of 120 mM-tetraethylammonium-methanesulphonate and 10 mM-Ca2+. The recording solution was made hypertonic by adding 350 mM-sucrose to avoid contraction. Two components of inward current in the absence of external Na+ were observed. Depolarization induced a fast-activated inward current of small amplitude in addition to the well-known slow, transient Ca2+ current (ICa,s). Both components of inward current persisted in the presence of tetrodotoxin. They practically disappeared on replacing external Ca2+ with Mg2+ and were blocked by millimolar additions of Cd2+ to the bath. Thus, the fast-activated component of inward current was also carried by Ca2+ (ICa,f). Neither ICa,f nor ICa,s were reduced by 5 microM-diltiazem. During 400 ms depolarizations ICa,f was detected at approximately -60 mV, 30 mV more negative than the membrane potentials at which ICa,s appeared. At about 0 mV the time constant for activation was 5 ms for ICa and 150 ms for ICa,s. ICa,f did not significantly decline during depolarizations up to 2s in duration at membrane potentials between -60 and -30 mV. ICa,f tended to disappear as a function of time on exposure to the hypertonic recording solution. Its maximum amplitude decreased from about -25 microA/cm2 during the first 5 min to about -5 microA/cm2 after 25 min while ICa.s remained practically unchanged (maximum peak amplitude of about -60 microA/cm2). These results indicate the existence of two types of voltage-dependent CA2+ channels in intact muscle fibres. The kinetic properties of fast-activated Ca2+ channels suggest that they significantly activate during a single twitch.

Action Potentials↗

PubMed1984

Saturation of calcium channels and surface charge effects in skeletal muscle fibres of the frog.

Voltage-clamp and current-clamp experiments were performed to study Ca2+ and Ba2+ permeation through Ca channels in intact twitch skeletal muscle fibres of the frog. Surface charge effects were taken into consideration. Ca2+ (ICa) or Ba2+ (IBa) currents, or Ca2+ and Ba2+ action potentials were recorded in the presence of external tetraethylammonium (TEA+) ions and by replacing C1- for CH3SO3-. To further block K+ outward currents, muscles were incubated in a K+-free, TEA+ and Cs+-containing solution prior to experiments. When 10 mM-Ca2+ was replaced by 10 mM-Ba2+, the I/V curve for the peak inward current shifted by 15-20 mV to more negative potentials and the maximal peak inward current increased from -39 +/- 2 mA cm-3 (5) to -51 +/- 3 mA cm-3 (7). The decay of ICa and IBa followed a simple exponential time course and became faster for large depolarizations. The overshoot of the action potentials changed 29 +/- 3 mV or 32 +/- 3 mV for a 10-fold change in the Ca2+ or Ba2+ concentrations respectively. Ca2+ action potentials were 15-20 mV larger than Ba2+ action potentials. The maximum rate of rise Vmax and the Ca2+ or Ba2+ conductance GC2+ during the plateau tend to saturate as divalent cation concentration was increased. The Michaelis constant (Km) values obtained were respectively: 5.6 and 6.0 mM for Ca2+ and 12.5 and 8.0 mM for Ba2+. When Ca2+ or Ba2+ concentrations were increased, the effective threshold of the inward current Theff and the membrane potential E* at Vmax shifted to more positive potentials along the voltage axis. These shifts were similar for Theff and E* and were more pronounced for Ca2+ than for Ba2+. Voltage shifts could be adequately quantified by the Gouy-Chapman theory with a density of surface charges near Ca channels of 0.20 e nm-2 and including a specific binding constant for Ca2+ of 45 +/- 4 m-1. The fractional increase of the Ca2+ and Ba2+ calculated concentrations at the membrane surface near the channel was smaller than the corresponding one in the bulk solution. This partially explained the reported saturation. Saturation was still present in the Vmax of GC2+ curves corrected for surface concentration. The corrected Km values for the Vmax data were 60 mM for Ca2+ and 350 mM for Ba2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1984

Calcium channel inactivation in frog (Rana pipiens and Rana moctezuma) skeletal muscle fibres.

The decay of the Ca2+ current (ICa) during a maintained depolarization was studied in intact twitch skeletal muscle fibres of Rana pipiens and Rana moctezuma with the three-micro-electrode voltage-clamp technique. ICa was recorded at 23 degrees C, after blocking K+ currents, in TEA methanesulphonate saline with 10 mM-Ca2+ made hypertonic by adding 350 mM-sucrose. In two-pulse experiments, ICa during the test pulse was reduced to about 80% (R. pipiens) or 50% (R. moctezuma) of the control value, without any detectable inward ICa during 7 s conditioning pre-pulses. The experimental points of the steady-state inactivation curve (h infinity) were fitted to h infinity = (1 + exp [Em - Vh)/kh]-1, where Em is the membrane potential and with Vh = -33 +/- 3 mV and kh = 6 +/- 1 mV for R. pipiens, and Vh = -44 +/- 3 mV and kh = 9.5 +/- 1.0 mV for R. moctezuma. The rate constant of decay for inactivated currents (range -8 to -47 mA cm-3) and for control currents (range -23 to -62 mA cm-3), was independent of ICa amplitude. The average rate constant of decay at 0 mV was 1.18 +/- 0.02 s-1 (66). These results indicate that in intact fibres under hypertonic solution ICa decay can be explained by a voltage-dependent inactivation process and not by depletion of tubular Ca2+. The absence of depletion could be due to a large fractional tubular volume or to the presence of a Ca2+ pump in the tubular system.

Animals↗

PubMed1983

Calcium-channel gating in frog skeletal muscle membrane: effect of temperature.

Voltage-clamp experiments using the three micro-electrode method were performed to study the temperature dependence of the calcium current ICa in intact twitch skeletal muscle fibres of the frog. Contraction was blocked by recording in hypertonic sucrose solutions. For depolarizations smaller than 0 mV the decay of the transient, slow, inward current, recorded in the presence of external tetraethylammonium (TEA+) and by replacing Cl- for CH3SO3-, followed a complex time course. For larger depolarizations, after the initial inward current, there was a prominent, slow, outward current which showed two phases: after reaching a peak (time to peak 1.0 sec, peak amplitude 20-50 microA/cm2 at 20 mV) it slowly declined to a steady level in about 2-3 sec at 23 degrees C. The inward current was greatly reduced or abolished by the adding of 2 mM-Cd2+ or by replacing external Ca2+ with Mg2+. The amplitude and time course of slow, outward currents were not obviously modified by replacing Ca2+ with Mg2+, having the two described phases. However, in the presence of Cd2+ the first transient phase of the outward current was not detected and only outward currents slowly increasing to a steady level were observed. Reliable ICa records were obtained by further blocking K+ outward currents by incubating the muscles in a K+-free TEA+- and Cs+-containing solution prior to experiments. Tubular space clamp was improved by recording ICa from small fibres with 20-30 microns radius. The decay phase of ICa under a maintained depolarization in incubated muscles was fitted by a single exponential. The corresponding rate constant determined between 12 and 24 degrees C strongly depended on temperature, as expected for a gating process. The values for the activation energy and the corresponding Q10 (calculated for a 10-20 degrees C transition) were respectively: 17.5 +/- 1.0 kcal/mole and 2.9 +/- 0.2 at 0 mV, and 18.0 +/- 1.5 kcal/mole and 3.0 +/- 0.3 at -20 mV. The activation phase of ICa, analysed following the m alpha h Hodgkin-Huxley kinetic model, showed a similar temperature dependence with a Q10 of 3.0 +/- 0.3. The peak amplitude of ICa and the limiting Ca2+ permeability had a lower Q10 value of about 1.6. For a given temperature the rate constant of decay was independent of ICa peak amplitude in disagreement with a current-dependent process (intratubular Ca2+ depletion or intracellular Ca2+ accumulation) for the decay of ICa. In conclusion, our results favour a gating process (inactivation) as the principal mechanism underlying the decay phase of ICa under a maintained depolarization.

Action Potentials↗

PubMed1982

External calcium and contractile activation during potassium contractures in twitch muscle fibres of the frog.

Effects of external Ca2+ concentration reduction on the amplitude and time course of K+ contractures were studied in single muscle fibres. The resting potential, effective resistance, threshold for the Na current, action potential and K+-induced depolarizations did not change when 1.8 mM Ca2+ was replaced by 3 mM Mg2+ (3--6 microM Ca2+). Identical results were obtained after the addition of 5 mM EGTA (less than or equal to 10(-9) M Ca2+; Ca-free saline). The rate of tension development during the initial phase of K+ contractures was independent of external Ca2+ while the amplitude, the duration, and the time constant of spontaneous relaxation decreased progressively as Ca2+ concentration was diminished. The activation curve shifted by 3--5 mV towards more positive potentials while the inactivation curve shifted by 16--18 mV in the opposite direction and both curves became steeper in Ca-free saline. External Ca2+ may play a role in excitation--contraction coupling during K contractures either via the inward Ca current or via specific interactions between external Ca2+ ions and the coupling mechanism or both.

Action Potentials↗

PubMed1981

Effects of external calcium reduction on the kinetics of potassium contractures in frog twitch muscle fibres.

1. The amplitude and time course of K contractures (Cl- constant) of single twitch muscle fibres of the frog have been analysed in three external Ca2+ concentrations. 2. The resting potential, effective resistance, threshold for the Na current and K-induced depolarizations were not modified by replacing 1.8 mM-Ca2+ by 3 mM-Mg2+ in absence (low-Ca saline: 3-6 micro M-Ca2+) or in the presence of 5 mM-EGTA (Ca-free saline: less than or equal to 10(-9) M-Ca2+). 3. The tension development during the initial phase of K contractures was independent of external Ca2+ while the amplitude, the duration and the time constant of spontaneous relaxation decreased progressively as Ca2+ concentration was diminished. 4. When the concentration of Mg2+ was increased to 5 mM in Ca-free saline K contractures were slower and smaller than those in 3 mM-Mg2+. 5. In Ca-free saline the activation curve (peak tension vs. logarithm of external K+ concentration) shifted by 3-5 mV towards more positive potentials while the inactivation curve (peak tension of the test contracture vs. logarithm of external K+ concentration during the conditioning period) shifted by 16-18 mV towards more negative potentials. Both curves became steeper in Ca-free saline. 6. The effects of external Ca2+ reduction were not modified by replacing all chloride for methanesulphonate. 7. Direct effects of external Ca2+ on excitation-contraction coupling during K contractures could involve the inward Ca current and/or specific interactions between external Ca2+ ions and the coupling mechanism.

Animals↗